Cytomegalovirus Surface Protein Complex for use in Vaccines and as a Drug Target

ABSTRACT

Immunogenic compositions and prophylactic or therapeutic vaccines for use in protecting and treating against human cytomegalovirus (CMV) are disclosed. Subunit vaccines comprising a human CMV protein complex comprising pUL128 or pUL130, and nucleic acid vaccines comprising at least one nucleic acid encoding a CMV protein complex comprising pUL128 or pUL130 are described. Also disclosed are therapeutic antibodies reactive against a CMV protein complex comprising pUL128 or pUL130, as well as methods for screening compounds that inhibit CMV infection of epithelial and endothelial cells, methods for immunizing a subject against CMV infection, methods for determining the capability of neutralizing antibodies to inhibit CMV infection of cell types other than fibroblasts, and methods of diminishing an CMV infection.

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 14/474,726,filed Sep. 2, 2014, which is a divisional of application Ser. No.13/409,987, filed Mar. 1, 2012, now U.S. Pat. No. 8,828,399, which is adivisional of application Ser. No. 12/766,611, filed Apr. 23, 2010, nowU.S. Pat. No. 8,173,362, which is a divisional of application Ser. No.11/810,578, filed Jun. 6, 2007, now U.S. Pat. No. 7,704,510, whichclaims benefit of U.S. Provisional Application No. 60/811,689, filedJun. 7, 2006, and U.S. Provisional Application No. 60/902,544, filedFeb. 20, 2007, the entire contents of each of which are incorporated byreference herein.

GOVERNMENT SUPPORT

This invention was made with government support under Grant Nos.CA85786, CA82396, AI54430, and GM71508 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

FIELD OF THE INVENTION

The invention relates generally to the fields of vaccine development,passive immunity and antiviral drug discovery. More specifically, theinvention relates to vaccines to cytomegalovirus, the development ofantibodies as therapeutic agents for treatment of cytomegalovirusinfections, and to screening assays for identification of molecules thatinhibit cytomegalovirus infectivity.

BACKGROUND OF THE INVENTION

Various publications, including patents, published applications,technical articles and scholarly articles are cited throughout thespecification. Each of these cited publications is incorporated byreference herein, in its entirety.

Cytomegalovirus (CMV) is a herpes virus classified as being a member ofthe beta subfamily of herpesviridae. According to the Centers forDisease Control and Prevention, CMV infection is found fairlyubiquitously in the human population, with an estimated 40-80% of theUnited States adult population infected. The virus is spread primarilythrough bodily fluids, and is frequently passed from pregnant mothers tothe fetus or newborn. In most individuals, CMV infection is latent,although virus activation can result in high fever, chills, fatigue,headaches, nausea, and splenomegaly.

Although most human CMV infections are asymptomatic, CMV infections inimmunocompromised individuals, such as newborns, HIV-positive patients,allogeneic transplant patients and cancer patients, can be particularlyproblematic. CMV infection in such individuals can cause severemorbidity, including pneumonia, hepatitis, encephalitis, colitis,uveitis, retinitis, blindness, and neuropathy, among other deleteriousconditions. In addition, CMV is a leading cause of birth defects (BrittW J et al. 1996, Fields Virology, 3rd ed. 2493-2523). At present, thereis no cure or preventive vaccine for CMV infection.

CMV infects various cells, including monocytes, macrophages, dendriticcells, neutrophils, endothelial cells, epithelial cells, fibroblasts,neurons, smooth muscle cells, hepatocytes, and stromal cells (Plachter Bet al. 1996, Adv. Virus Res. 46:195-261). Infection of epithelial cellsis significant because epithelial cells facilitate the spread of thevirus within the host (Britt & Alford, 1996, supra). Infection ofendothelial cells is significant because such cells are believed to besites of human CMV persistence and latency, because endothelial cellsare believed to be a gateway to leukocyte infection, because endothelialcells may facilitate mother to fetus/neonate transmission, and becauseinfection of vascular endothelial cells is believed to contribute tovarious vascular pathologies, among other things (Jarvis M A et al.2002, Curr. Opin. Microbiol. 5:403-7; Gerna G et al. 2002, J. Virol.74:5629-38; Hengel et al. 2000, Trends Microbiol. 8:294-6; and, PatroneM et al. 2005, J. Virol. 79:8361-73).

In immunocompromised individuals, CMV infects multiple organ systems,replicating in all major cell types. Although clinical isolatesreplicate in a variety of cell types, laboratory strains, such as AD169(Elek, S. D. & Stern, H. 1974, Lancet 1, 1-5) and Towne (Plotkin, S. A.,et al. 1975, Infect. Immun. 12, 521-527) replicate almost exclusively infibroblasts (Hahn G et al. 2004, J. Virol. 78:10023-33). The restrictionin tropism, which results from serial passage of the virus infibroblasts, is a marker of attenuation (Gerna et al. 2002, supra).Mutations causing the loss of epithelial cell, endothelial cell,polymorphonuclear leukocyte, and dendritic cell tropism in human CMVlaboratory strains have been mapped to three open reading frames (ORFs):UL128, UL130, and UL131 (Hahn et al. 2004, supra; Gerna, G., et al.2005, J. Gen. Virol. 86, 275-284). Mutation of any one of these ORFs inthe FIX clinical isolate of human CMV blocked endothelial cell tropism(Hahn et al. 2004, supra).

CMV particles contain three major glycoprotein complexes, all of whichare required for human CMV infectivity. The gCI complex includes twomolecules of the UL55-coded gB. Each 160-kDa monomer is cleaved togenerate a 116-kDa surface unit linked by disulfide bonds to a 55-kDatransmembrane component. Some antibodies immunospecific for gB inhibitthe attachment of virions to cells, whereas others block the fusion ofinfected cells, suggesting that the protein might execute multiplefunctions at the start of infection. Several cellular membrane proteinsinteract with gB, and these interactions likely facilitate entry andactivate cellular signaling pathways. The gCII complex contains the UL100-coded gM and UL73-coded gN, and it is the most abundant of theglycoprotein complexes. The complex binds to heparan sulfateproteoglycans, suggesting it might contribute to the initial interactionof the virion with the cell surface. It also could perform a structuralrole during virion assembly/envelopment, similar to the gM-gN complexfound in some α-herpesviruses. The gCIII complex is comprised ofUL75-coded gH, UL115-coded gL, and UL74-coded gO. All knownherpesviruses encode gH-gL heterodimers (Spear, P. G. & Longnecker, R.2003, J. Virol. 77, 10179-10185), which mediate fusion of the virionenvelope with the cell membrane. Antibodies immunospecific for human CMVgH do not affect virus attachment but block penetration and cell-to-cellspread (Rasmussen, L. E. et al. 1984, Proc. Natl. Acad. Sci. USA 81,876-880; Keay, S. & Baldwin, B. 1991, J. Virol. 65, 5124-5128).Expression of gH-gL in the absence of infection was sufficient to inducesyncytia, and inclusion of gO in the assay did not enhance or block thefusion (Kinzler, E. R. & Compton, T. 2005, J. Virol. 79, 7827-7837). AgO-deficient mutant of AD169 shows a significant growth defect (Hobom,U. et al. 2000, J. Virol. 74, 7720-7729). Recently, it was reported thatgH binds to integrin a v133 (Wang, X. et al. 2005, Nat. Med. 11,515-521). However, the proteins encoded by UL131-UL128 heretofore havenot been reported to be associated with any of the viral glyocoproteins.

There is a need for a CMV vaccine, and for effective means to controlthe spread and activation of the virus, particularly inimmunocompromised individuals and pregnant women. There is also a needfor methods to screen for antiviral compounds that inhibitcytomegalovirus infectivity.

SUMMARY OF THE INVENTION

One aspect of the invention features an immunogenic compositioncomprising a pharmaceutically acceptable carrier and a complex ofcytomegalovirus (CMV) proteins comprising pUL128 or pUL130 and,optionally, at least one other virus or cellular constituent of a CMVvirion complex. In various embodiments, the CMV proteins are fromprimate CMV, such as human, chimpanzee or rhesus monkey CMV.

In various embodiments, the other virus or cellular constituent of thevirion complex can be one or more of pUL131, gH, gL, or gB. Forinstance, the complex can comprise pUL128 and pUL130, or it can comprisepUL128 alone or pUL130 alone. Or, the complex can comprise pUL128 and gHor gL, or pUL130 and gH or gL, or pUL128, pUL130 and gH or gL, or allfour proteins. Alternatively, for instance, the complex can comprisepUL128, gH and gL or pUL30, gH and gL.

In one embodiment, multiple fragments of pUL128 are linked into onepolypeptide chain. Alternatively, multiple fragments of pUL130 arelinked into one polypeptide chain. In another embodiment, multiplefragments of pUL128 and pUL130 are linked into one polypeptide chain, ormultiple fragments of pUL128, pUL130 and gH or gL are linked into onepolypeptide chain. In yet another embodiment, multiple fragments ofpUL128 and glycoprotein B are linked into one polypeptide chain, ormultiple fragments of pUL130 and glycoprotein B are linked into onepolypeptide chain. In another embodiment wherein multiple fragments ofpUL128, pUL130 and glycoprotein B are linked into one polypeptide chain.

In this aspect of the invention, the complex can be produced byexpression of one or more polynucleotides encoding the CMV proteins. Forinstance, the complex can be produced by expression of a CMV genomeencoding an attenuated CMV, wherein the attenuation does not affectformation of the complex.

Another aspect of the invention features a subunit vaccine comprising apharmaceutically acceptable carrier and at least one cytomegalovirus(CMV) protein or fragment thereof, selected from pUL128, pUL130, or acomplex that includes pUL128 or pUL130, and, optionally, at least oneother virus or cellular constituent of a CMV virion complex, wherein thevaccine induces an immune response against CMV in a recipient. Invarious embodiments, the CMV protein or fragment thereof is from primateCMV, such as a human, chimpanzee or rhesus monkey CMV. The subunitvaccine can further comprise an adjuvant.

In certain embodiments, at least one protein of the complex is coupledto a carrier protein. Suitable carrier proteins include, but are notlimited to, albumin, ovalbumin, Pseudomonas exotoxin, tetanus toxin,ricin toxin, diphtheria toxin, cholera toxin, heat labile enterotoxin,keyhole lympet hemocyanin, epidermal growth factor, fibroblast growthfactor, transferring, platelet-derived growth factor, poly-L-lysine,poly-L-glutamine, or mannose-6-phosphate.

In one embodiment, the protein, protein fragment or complex is expressedon the surface of an attenuated CMV virus particle. In anotherembodiment, the protein or fragment thereof is fused to one or moreother proteins or fragments thereof present on the surface of the CMVvirus particle. In another embodiment, the protein or fragment thereofis fused to at least one non-human CMV protein modified for expressionon the surface of the human CMV virus particle.

Another aspect of the invention features a nucleic acid vaccinecomprising a pharmaceutically acceptable carrier and a vector comprisingat least one nucleic acid molecule encoding a CMV protein or fragmentthereof, selected from pUL128, pUL130, or a complex that includes pUL128or pUL130, wherein the at least one nucleic acid molecule is expressedin a vaccine recipient, and wherein the expression product induces animmune response against CMV in the recipient.

The nucleic acid vaccine can be constructed to express one or moreproteins involved in the pUL128-pUL130-containing complex, and/or othervirion proteins. In one embodiment, the CMV proteins are contained on anon-CMV vector. For instance, non-CMV vectors can express pUL128, orpUL130, or both pUL128 and pUL130. Or, non-CMV vectors can expresspUL128, pUL130 and gH, gL or both gH and gL. In other embodiments, thenucleic acid vaccine can comprise a non-CMV vector that expresses one ormore fragments of one or more of pUL128, pUL130, pUL131, gH, gL, or gB.In particular embodiments, two or more of such fragments are expressedon a single polypeptide.

Another aspect of the invention features antibodies or epitope-bindingfragments thereof, which specifically bind to a virus-coded protein froma CMV virion complex that includes pUL128 or pUL130, wherein theantibodies or epitope-binding fragments thereof inhibit binding of theCMV virion complex to a cellular receptor, or CMV infection of a cell,or both. In various embodiments, the virion is from primate CMV, e.g.,human, chimpanzee or rhesus monkey CMV.

The antibodies can be monoclonal antibodies or single-chain antibodiesproduced by recombinant DNA methods. In certain embodiments, they arehuman or humanized antibodies.

In certain embodiments, the antibodies specifically bind to pUL128. Inone embodiment, the antibodies have equal or greater binding affinityfor pUL128 than polyclonal antibody of antiserum having ATCC AccessionNo. PTA-8474. Exemplary antibodies of this type are polyclonalantibodies of antiserum having ATCC Accession No. PTA-8474. In anotherembodiment, the antibodies have equal or greater binding affinity forpUL128 than monoclonal antibodies produced by a hybridoma cell linehaving ATCC Accession No. PTA-8473. In another embodiment, theantibodies compete for binding to an epitope on pUL128 recognized bymonoclonal antibodies produced by a hybridoma cell line having ATCCAccession No. PTA-8473. The antibodies can bind to the same epitope onpUL128 as do the monoclonal antibodies produced by the hybridoma cellline having ATCC Accession No. PTA-8473. Exemplary monoclonal antibodiesof this type are monoclonal antibodies produced by a hybridoma cell linehaving ATCC Accession No. PTA-8473. Another embodiment featuresneutralizing binding partner of a CMV virion complex comprising pUL128,which comprises one or more virion binding sequences having 70% orgreater identity to one or more complementarity determining regions(CDR) present in the monoclonal antibodies produced by a hybridoma cellline having ATCC Accession No. PTA-8473.

In certain embodiments, the antibodies specifically bind to pUL130. Inone embodiment, the antibodies have equal or greater binding affinityfor pUL130 than monoclonal antibodies produced by a hybridoma cell linehaving ATCC Accession No. PTA-8472. In another embodiment, theantibodies compete for binding to an epitope on pUL130 recognized bymonoclonal antibodies produced by a hybridoma cell line having ATCCAccession No. PTA-8472. The antibodies can bind to the same epitope onpUL130 as do the monoclonal antibodies produced by the hybridoma cellline having ATCC Accession No. PTA-8472. Exemplary monoclonal antibodiesof this type are monoclonal antibodies produced by a hybridoma cell linehaving ATCC Accession No. PTA-8472. Another embodiment featuresneutralizing binding partner of a CMV virion complex comprising pUL130,which comprises one or more virion binding sequences having 70% orgreater identity to one or more complementarity determining regions(CDR) present in the monoclonal antibodies produced by a hybridoma cellline having ATCC Accession No. PTA-8472.

Any of the foregoing antibodies can further comprise glycosylation thathas been modulated by expression in yeast cells that have beenengineered to add glycan structures to proteins. Further, any of theforegoing antibodies can be formulated into a pharmaceuticalcomposition.

In various embodiments, the foregoing antibodies are produced byexposing an immunocompetent subject to a CMV virion complex comprisingpUL128 or pUL130 and at least one other virus or cellular constituent ofthe virion complex, wherein the antibodies are immunospecific for theCMV virion complex comprising pUL128 or pUL130, but are notimmunospecific for any other CMV virion complex. Such antibodies can bepolyclonal antibodies having components that bind to pUL128 or pUL130.In a particular embodiment, they are polyclonal antibodies havingcomponents that bind to pUL128 and components that bind to pUL130,wherein the antibodies are capable of binding at least twice as muchpUL130 as pUL128.

Another aspect of the invention features a method of inhibiting CMVinfection of endothelial or epithelial cells, comprising inhibitingbinding of a CMV virion complex comprising pUL128 or pUL130 to thecells, thereby inhibiting the CMV infection. In one embodiment, bindinginhibition is accomplished by treating the cells with an antibodyimmunospecific for the CMV virion complex, particularly for pUL128 orpUL130. The method can be practiced on cultured cells or in situ incells within a living organism.

Another aspect of the invention features a method for screeningcompounds for the ability to inhibit entry of CMV into host cells, whichcomprises: (a) exposing host cells, in the presence or absence of a testcompound, to one or more cellular receptors of host cells to CMV virionsor a component thereof selected from (i) pUL128 or a fragment thereof,(ii) pUL130 or fragment thereof, or (iii) a complex that includes pUL128or pUL130, and, optionally, at least one other virus or cellularconstituent of a CMV virion complex; and (b) determining if the testcompound interferes with binding of the CMV virions or component thereofto the host cells or cellular receptors, wherein the interfering of thebinding is indicative that the test compound is capable of inhibitingthe entry of the CMV into the host cells.

In certain embodiments of the method, the host cells are epithelialcells or endothelial cells. In one embodiment, the cellular receptorsare disposed within a membrane fragment. The cellular receptors can beaffixed to a solid support. In one embodiment, the CMV component ispUL128 or a fragment thereof, or pUL130 or a fragment thereof. The CMVcomponent also can be affixed to a solid support. The CMV virions can beproduced by expressing a virion encoding polynucleotide in cellstransfected with a vector containing the polynucleotide. In an exemplaryembodiment, the vector is BADrUL131. The test compound can be abiomolecule, organic chemical, inorganic chemical, or a fragment,analog, homolog, conjugate, or derivatives thereof.

In another embodiment, a selected test compound determined by theforegoing method to be capable of interfering with the binding of CMV orcomponents thereof to the host cells or cellular receptors is subjectedto a secondary screen comprising: (a) exposing the host cells to CMVvirions in the presence or absence of the selected test compound; and(b) determining if the selected test compound inhibits one or more of(i) production of CMV proteins within the host cells; (ii) a cytopathiceffect of CMV infection; or (iii) spread of virus proteins from cell tocell, the inhibition being further indicative that the test compound iscapable of inhibiting the CMV infection.

Another aspect of the invention features a method of screening compoundsfor their ability to neutralize human CMV infectivity of endothelial orepithelial cells. The method comprises: (a) exposing the epithelial orendothelial cells to CMV virions comprising a virion complex thatincludes pUL128 or pUL130, in the presence or absence of a testcompound; and (b) determining if the test compound inhibits entry of theCMV into the host cells, the inhibition being indicative that the testcompound is able to neutralize human CMV infectivity of the endothelialor epithelial cells. In particular embodiments, the test compound is anantibody or epitope-binding fragment thereof, or a neutralizing bindingpartner of a CMV virion complex comprising pUL130 or pUL128. The CMVvirions can be produced by expressing a virion encoding polynucleotidein cells transfected with a vector containing the polynucleotide. In oneembodiment, the vector contains a genome of a clinical isolate of CMV.In another embodiment, the vector contains a genome of a laboratorystrain of CMV that comprises, or that has been engineered to comprise afunctional UL131-128 locus. In an exemplary embodiment, the vector isBADrUL131.

Another aspect of the invention features a method of immunizing apatient against CMV infection by administering to the patient animmunogenic composition comprising a pharmaceutically acceptable carrierand a complex of cytomegalovirus (CMV) proteins comprising pUL128 orpUL130 and, optionally, at least one other virus or cellular constituentof a CMV virion complex, under conditions permitting the patient todevelop an immune response to the immunogenic composition.

Another aspect of the invention features a method of immunizing apatient against CMV infection by administering to the patient a subunitvaccine comprising a pharmaceutically acceptable carrier and at leastone cytomegalovirus (CMV) protein or fragment thereof, selected frompUL128, pUL130, or a complex that includes pUL128 or pUL130, and,optionally, at least one other virus or cellular constituent of a CMVvirion complex, under conditions permitting the patient to develop animmune response to the subunit vaccine.

Yet another aspect of the invention features a method of immunizing apatient against CMV infection by administering to the patient a nucleicacid vaccine comprising a pharmaceutically acceptable carrier and avector comprising at least one nucleic acid molecule encoding a CMVprotein or fragment thereof, selected from pUL128, pUL130, or a complexthat includes pUL128 or pUL130, wherein the at least one nucleic acidmolecule is expressed in the patient, under conditions permitting thepatient to develop an immune response to the proteins encoded by thenucleic acid vaccine.

Still another aspect of the invention features a method of diminishing aCMV infection in a patient, comprising administering to the patientantibodies or epitope-binding fragments thereof, which specifically bindto a virus-coded protein in a CMV virion complex that includes pUL128 orpUL130, wherein the antibodies or epitope-binding fragments thereofinhibit binding of the human CMV virion complex to a cellular receptor,or CMV infection of a cell, or both, thereby diminishing the CMVinfection in the patient.

Other features and advantages of the invention will be understood byreference to the drawings, detailed description and examples thatfollow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the human CMV UL131-128 locus. (A) Diagram of the locus.The positions of transcriptional start sites and poly(A) cleavage sitesare indicated. Solid boxes represent the sequence of wild-type ORFs. Thelocation of the point mutation in the AD169 UL131 gene is indicated, andthe portion of UL131 that is not expressed is designated by an open box.(B) Amino acid sequence of the N-terminal domain of UL131 in tworepaired AD169 derivatives, BADrUL131-Y4 (SEQ ID NO: 1) and BADrUL131-C4(SEQ ID NO:2, and human CMV variant strains, pTNUL131 (SEQ ID NO:3),pTLUL131 (SEQ ID NO:4), pFXUL131 (SEQ ID NO:5), and pTRUL131 (SEQ IDNO:6). The likely signal peptide cleavage sites are indicated.

FIG. 2 shows that pUL128 and pUL130 form a complex with gH. MRC-5 cellswere infected with BADd1UL131-128, BADwt, or BADrUL131. Seventy-twohours after infection, cells were radiolabeled for 1 h, and chased for20 or 120 min. Proteins were immunoprecipitated from cell lysates andanalyzed by SDS PAGE, followed by autoradiography. Immunoprecipitationsused anti-gB 7-17 (A), anti-gM IMP91-3/1 (B), anti-gH 14-4b (C), oranti-gH 14-4b, followed by anti-gO, anti-pUL130 3C5, or anti-pUL128R551A (D). The positions at which marker proteins migrated areidentified by their molecular weights (M.W.) in kilodaltons.

FIG. 3 shows that pUL128-pUL130 and gO form separate complexes with gH.MRC-5 cells were infected with BADd1UL131-128, BADwt, or BADrUL131.(A-C) Cells were radiolabeled for 1 h and chased for 20 or 120 minbeginning at 72 h post infection. Proteins were immunoprecipitated fromcell lysates and analyzed by SDS PAGE followed by autoradiography.Immunoprecipitations used anti-gO (A), anti-pUL128 4B10 (B), oranti-pUL130 3E3 (C). (D) Displays combined immunoprecipitation andWestern blot assays of pUL128-interacting proteins. Cells were lysed at72 h post infection, and extracts were subjected to immunoprecipitationwith anti-pUL128 R551A antibody. The precipitated proteins wereseparated by SDS PAGE and analyzed by Western blotting with anti-gHAP86, anti-pUL130 3C3, or anti-gO antibodies. The positions at whichmarker proteins migrated are identified by their molecular weights(M.W.) in kilodaltons. Antibody heavy (HC) and light chains (LC) aredesignated.

FIG. 4 shows that pUL128 and pUL130 are in virions. (A) BADwt andBADrUL131 virion proteins were analyzed by Western blotting usinganti-gH AP86, anti-pUL130 3C5, or anti-pUL128 4B10 antibodies. (B)Virion proteins were immunoprecipitated with anti-pUL128 R551A andanalyzed by Western blotting using anti-gH AP86 or anti-pUL130 3C5antibodies. The positions at which marker proteins migrated areidentified by their molecular weights (M.W.) in kilodaltons. Antibodyheavy chains (HC) are designated.

FIG. 5 shows the characterization of gH-gL complexes. (A) Disulfidelinkage of pUL128 with gH-gL. Purified BADrUL131 proteins in buffer withor without 2-mercaptoethanol (13-ME) were subjected SDS PAGE 12% (leftpanel) or 4-20% (right panel), and analyzed by Western blotting usinganti-pUL130 3C5 (left panel) or anti-pUL128 4B10 (right panel)antibodies. (B) Comparison of complexes in BADwt and BADrUL131 virions.Virion proteins were separated by reducing or nonreducing PAGE (8%), andanalyzed by Western blotting using anti-gO (left panel), anti-gH AP86(center panel), or anti-pUL128 4B10 (Right) antibody. *=monomeric gH;The filled diamond and circle identify monomeric forms of gO. Thepositions at which marker proteins migrated are identified by theirmolecular weights (M.W.) in kilodaltons.

FIG. 6 shows the neutralization of human CMV infectivity in ARPE-19epithelial cells, HUVEC endothelial cells, and MRC-5 fibroblasts.BADrUL131 (A) or BFXwt (B) were incubated with various concentrations ofanti-pUL130 3C5 or 3E3 or anti-pUL128 R551A antibodies, and residualinfectivity was determined on the different cell types.

FIG. 7 shows that CD46-specific antibody significantly diminishes humanCMV infection of ARPE-19 cells but not MRC-5 fibroblasts. Inhibition ofinfection in ARPE-19 cells increased with increasing concentrations ofanti-CD46 antibody, consistent with a dose-dependent effect. Infectionwas monitored by quantifying the number of cells expressing the 1E1protein encoded by human CMV.

FIG. 8 shows that CYTOGAM® contains antibodies that react with UL130protein. GST fusion proteins containing pUL128 (aa 28-171), pUL130 (aa37-133), pUL131 (aa 28-129) or unfused GST were produce in E. coli,partially purified, resolved by electrophoresis (12% SDS-PAGE), andtransferred to a nitrocellulose membrane. After blocking with skim milk,the blot was probed with a 1:5000 dilution of CYTOGAM®. The proteinsreacting with CYTOGAM® were then detected by using HRP conjugatedanti-human IgG and enhanced chemiluminescence. The GST fusion proteinpreparations include degraded species.

FIG. 9 shows isolation of antibody subpopulations from CYTOGAM®. MRC-5cells were transfected with a gB expression plasmid or infected withBADd1UL(128-131) or BADrUL131 at a multiplicity of 1 pfu/cell. The cellswere fixed with 4% paraformaldehyde at 72 h post transfection orinfection. CYTOGAM® was diluted to 10 mg/ml and sequentially absorbed tothe MRC-5 cells with each adsorption for 2 h at room temperature. Afteradsorption, the cells were washed three times with DPBS. Boundantibodies were eluted from plates using 0.4 M acetic acid for 5 min atroom temperature, and the eluate was immediately neutralized using asaturated Tris solution to a final pH of 7.0 to 8.0. Eluted antibodieswere dialyzed, concentrated in dialysis tubing by dehydration usingpolyethylene glycol powder, and stored at 4° C.

FIG. 10 shows the relative neutralizing activity of gB versuspUL128/pUL130/pUL131 antibodies isolated from CYTOGAM®. Equal amounts ofgB ab or pUL128/130/131 antibody were serially diluted and incubatedwith ˜100 pfu of BADrUL131 for 1 h at room temperature. Afterincubation, solutions were added to ARPE-19 epithelial cells in 96-wellmicrotiter plates for 2 h at 37° C. All samples were tested induplicate. After the virus solutions were removed, cells were incubatedfor another 20 h in fresh medium, then washed, fixed withparaformadehyde, stained with anti-IE72 monoclonal antibody 12B10 andAlexa546 conjugated anti-mouse IgG secondary antibodies. The number offluorescence cells was compared to the number of cells infected by virusin medium lacking antibody to calculate percent residual infectivity.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Various terms relating to the methods and other aspects of the presentinvention are used throughout the specification and claims. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which the invention pertains. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice for testing of the present invention, the preferred materialsand methods are described herein. In describing and claiming the presentinvention, the following terminology will be used.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting. As used in this specification and the appended claims,the singular forms “a”, “an” and “the” include plural referents unlessthe content clearly dictates otherwise. Thus, for example, reference to“a cell” includes a combination of two or more cells, and the like.

“Antibody” or “immunoglobulin” is used broadly to refer to both antibodymolecules and a variety of antibody-derived molecules and includes anymember of a group of glycoproteins occurring in higher mammals that aremajor components of the immune system. The term “antibody” is used inthe broadest sense and specifically covers monoclonal antibodies,polyclonal antibodies, antibody compositions with polyepitopicspecificity, bispecific antibodies, diabodies, and single-chainmolecules, as well as antibody fragments (e.g., Fab, F(ab′)2, and Fv),so long as they exhibit the desired biological activity. Animmunoglobulin molecule includes antigen binding domains, which eachinclude the light chains and the end-terminal portion of the heavychain, and the Fc region, which is necessary for a variety of functions,such as complement fixation. There are five classes of immunoglobulinswherein the primary structure of the heavy chain, in the Fc region,determines the immunoglobulin class. Specifically, the alpha, delta,epsilon, gamma, and mu chains correspond to IgA, IgD, IgE, IgG and IgM,respectively. Immunoglobulin and antibody are deemed to include allsubclasses of alpha, delta, epsilon, gamma, and mu and also refer to anynatural (e.g., IgA and IgM) or synthetic multimers of the four-chainimmunoglobulin structure. Antibodies non-covalently, specifically, andreversibly bind an antigen. The antigen binding activity is found in theV (variable) region of the antibody whereas the complement fixing and Igreceptor binding activity is found in the C region. There are structuralconstraints on the amount of sequence variation allowed in the V region.In fact the variation is mostly restricted to three regions within theN-terminal domain of both the heavy (H) and light (L) chains. In the3-dimensional structure these regions form loops at the surface of theantibody molecule and these provide the binding surface between antibodyand antigen. Because these regions determine the ‘fit’ between antibodyand antigen they are referred to as the “complementarity determiningregions” or “CDRs”.

A “monoclonal antibody” is an antibody obtained from a population ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that can be present in minor amounts. For example,monoclonal antibodies may be produced by a single clone ofantibody-producing cells. Unlike polyclonal antibodies, monoclonalantibodies are monospecific (e.g., specific for a single epitope). Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. Screening assays to determine binding specificity ofan antibody are well known and routinely practiced in the art. For acomprehensive discussion of such assays, see Harlow et al. (Eds.),Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; ColdSpring Harbor, N.Y. (1988), Chapter 6.

The term “neutralizing antibody” refers to a form of antibody thatinteracts with an infectious agent, such as a virus, and reduces orinhibits its ability to infect host cells.

“Biomolecules” include proteins, polypeptides, nucleic acids, lipids,polysaccharides, monosaccharides, and all fragments, analogs, homologs,conjugates, and derivatives thereof.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of +20% or +10%, more preferably +5%, even more preferably+1%, and still more preferably +0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

A “coding region” of a gene consists of the nucleotide residues of thecoding strand of the gene and the nucleotides of the non-coding strandof the gene which are homologous with or complementary to, respectively,the coding region of an mRNA molecule which is produced by transcriptionof the gene.

A “coding region” of an mRNA molecule also consists of the nucleotideresidues of the mRNA molecule which are matched with an anti-codonregion of a transfer RNA molecule during translation of the mRNAmolecule or which encode a stop codon. The coding region may thusinclude nucleotide residues corresponding to amino acid residues whichare not present in the mature protein encoded by the mRNA molecule(e.g., amino acid residues in a protein export signal sequence).

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA. Unless otherwise specified, a “nucleotide sequenceencoding an amino acid sequence” includes all nucleotide sequences thatare degenerate versions of each other and that encode the same aminoacid sequence. Nucleotide sequences that encode proteins and RNA mayinclude introns.

“Effective amount” or “therapeutically effective amount” are usedinterchangeably herein, and refer to an amount of a compound,formulation, material, or composition, as described herein effective toachieve a particular biological result. Such results may include, butare not limited to, the inhibition of virus infection as determined byany means suitable in the art.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system. “Exogenous” refers to anymaterial introduced from or produced outside an organism, cell, tissueor system.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

As used herein, the term “fragment,” as applied to a nucleic acid,refers to a subsequence of a larger nucleic acid. A “fragment” of anucleic acid can be at least about 15 nucleotides in length; forexample, at least about 50 nucleotides to about 100 nucleotides; atleast about 100 to about 500 nucleotides, at least about 500 to about1000 nucleotides, at least about 1000 nucleotides to about 1500nucleotides; or about 1500 nucleotides to about 2500 nucleotides; orabout 2500 nucleotides (and any integer value in between).

As used herein, the term “fragment,” as applied to a protein or peptide,refers to a subsequence of a larger protein or peptide. A “fragment” ofa protein or peptide can be at least about 10 amino acids in length(e.g., as for a single linear epitope); for example at least about 20amino acids in length; at least about 50 amino acids in length; at leastabout 100 amino acids in length, at least about 200 amino acids inlength, at least about 300 amino acids in length, and at least about 400amino acids in length (and any integer value in between).

“Homologous, homology” or “identical, identity” as used herein, refersto the subunit sequence identity between two polymeric molecules, e.g.,between two nucleic acid molecules, such as, two DNA molecules or twoRNA molecules, or between two polypeptide molecules. When a subunitposition in both of the two molecules is occupied by the same monomericsubunit; e.g., if a position in each of two DNA molecules is occupied byadenine, then they are homologous at that position. The homology betweentwo sequences is a direct function of the number of matching orhomologous positions; e.g., if half (e.g., five positions in a polymerten subunits in length) of the positions in two sequences arehomologous, the two sequences are 50% homologous; if 90% of thepositions (e.g., 9 of 10), are matched or homologous, the two sequencesare 90% homologous. By way of example, the DNA sequences 3′ATTGCC5′ and3′TATGGC are 50% homologous.

As used herein, “immunization” or “vaccination” is intended forprophylactic or therapeutic immunization or vaccination. “Therapeuticvaccination” is meant for vaccination of a patient with CMV infection.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell. Unless it is particularly specifiedotherwise herein, the proteins, virion complexes, antibodies and otherbiological molecules forming the subject matter of the present inventionare isolated, or can be isolated.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, that can be infected with CMV. In certainnon-limiting embodiments, the patient, subject or individual is a human.

“Parenteral” administration of an immunogenic composition includes,e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning and amplification technology,and the like, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

“Pharmaceutically acceptable” refers to those properties and/orsubstances which are acceptable to the patient from apharmacological/toxicological point of view and to the manufacturingpharmaceutical chemist from a physical/chemical point of view regardingcomposition, formulation, stability, patient acceptance andbioavailability. “Pharmaceutically acceptable carrier” refers to amedium that does not interfere with the effectiveness of the biologicalactivity of the active ingredient(s) and is not toxic to the host towhich it is administered.

As used herein, “test compound” refers to any purified molecule,substantially purified molecule, molecules that are one or morecomponents of a mixture of compounds, or a mixture of a compound withany other material that can be analyzed using the methods of the presentinvention. Test compounds can be organic or inorganic chemicals, orbiomolecules, and all fragments, analogs, homologs, conjugates, andderivatives thereof. Biomolecules include proteins, polypeptides,nucleic acids, lipids, polysaccharides, and all fragments, analogs,homologs, conjugates, and derivatives thereof. Test compounds can be ofnatural or synthetic origin, and can be isolated or purified from theirnaturally occurring sources, or can be synthesized de novo. Testcompounds can be defined in terms of structure or composition, or can beundefined. The compound can be an isolated product of unknown structure,a mixture of several known products, or an undefined compositioncomprising one or more compounds. Examples of undefined compositionsinclude cell and tissue extracts, growth medium in which prokaryotic,eukaryotic, and archaebacterial cells have been cultured, fermentationbroths, protein expression libraries, and the like.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state associated with CMV infection.

The term “treatment” as used within the context of the present inventionis meant to include therapeutic treatment as well as prophylactic, orsuppressive measures for the disease or disorder. Thus, for example, theterm treatment includes the administration of an agent prior to orfollowing the onset of a disease or disorder thereby preventing orremoving all signs of the disease or disorder. As another example,administration of the agent after clinical manifestation of the diseaseto combat the symptoms of the disease comprises “treatment” of thedisease. This includes for instance, prevention of CMV propagation touninfected cells of an organism. The phrase “diminishing CMV infection”is sometimes used herein to refer to a treatment method that involvesreducing the level of infection in a patient infected with CMV, asdetermined by means familiar to the clinician.

“Variant” as the term is used herein, is a nucleic acid sequence or apeptide sequence that differs in sequence from a reference nucleic acidsequence or peptide sequence respectively, but retains essentialproperties of the reference molecule. Changes in the sequence of anucleic acid variant may not alter the amino acid sequence of a peptideencoded by the reference nucleic acid, or may result in amino acidsubstitutions, additions, deletions, fusions and truncations. Changes inthe sequence of peptide variants are typically limited or conservative,so that the sequences of the reference peptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference peptide can differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A variant of anucleic acid or peptide can be a naturally occurring such as an allelicvariant, or can be a variant that is not known to occur naturally.Non-naturally occurring variants of nucleic acids and peptides may bemade by mutagenesis techniques or by direct synthesis.

A “vector” is a replicon, such as plasmids, phagemids, cosmids,baculoviruses, bacmids, bacterial artificial chromosomes (BACs), yeastartificial chromosomes (YACs), as well as other bacterial, yeast andviral vectors, to which another nucleic acid segment may be operablyinserted so as to bring about the replication or expression of thesegment. “Expression vector” refers to a vector comprising expressioncontrol sequences operatively linked to a nucleotide sequence to beexpressed. An expression vector comprises sufficient cis-acting elementsfor expression; other elements for expression can be supplied by thehost cell or in an in vitro expression system. Expression vectorsinclude all those known in the art, such as cosmids, plasmids (e.g.,naked or contained in liposomes) and viruses (e.g., lentiviruses,retroviruses, adenoviruses, and adeno-associated viruses) thatincorporate the recombinant polynucleotide.

Cytomegalovirus replicates in many different cell types, includingepithelial cells, endothelial cells, and fibroblasts. However,laboratory strains of the virus, many of which were developed asattenuated vaccine candidates by serial passage in fibroblasts, havelost the ability to infect epithelial and endothelial cells. Theirgrowth is restricted primarily to fibroblasts, due to mutations in theUL131-UL128 locus, which is comprised of the UL131 (also referred to asUL131A by some investigators), UL130 and UL128 genes. Earlier workdemonstrated that the UL131-UL128 locus is a primary determinant ofhuman CMV endothelial cell host range, and it has been determined by thepresent inventors that a functional UL131-128 locus is required forepithelial cell tropism as well. However, the nature of this functionwas undetermined, and indeed it was speculated that one or more of theUL131-UL128 gene products was a secreted cytokine (Akter P C et al.,2003, J. Gen. Virol. 84, 1117-1122; Hahn et al., 2004, supra).

It has now been demonstrated in accordance with the present inventionthat two products of the UL131-UL128 locus, pUL130 and pUL128, form acomplex with gH and gL, but not gO. The AD169 laboratory strain, whichlacks a functional UL131 protein, produces virions containing only thegH-gL-gO complex. An epithelial and endothelial cell tropic AD169variant in which the UL131 ORF has been repaired, termed BADrUL131,produces virions that carry both gH-gL-gO and gH-gL-pUL128-pUL130complexes. Antibodies against pUL130 or pUL1128 block infection ofepithelial and endothelial cells by BADrUL131 and the fusion-inducingfactor X (FIX) clinical human cytomegalovirus isolate but do not affectthe efficiency with which fibroblasts are infected.

It has also been discovered in accordance with the present inventionthat the cell surface antigen CD46 is a receptor by which CMV gainsentry into epithelial and endothelial cells. As described in greaterdetail herein, monoclonal antibody specific for CD46 was shown to blockCMV infectivity into epithelial and endothelial cells in a concentrationdependent manner. Accordingly, CD46 is a new cellular target fordevelopment of antiviral agents.

Thus, one aspect of the invention features immunogenic compositions andvaccines for the prevention or treatment of CMV infection, and methodsof immunizing an individual using such compositions. Such vaccinestarget the interaction between CMV and its cellular receptors,particularly through the virion membrane complex that includes gH, gL,pUL128, pUL130, possibly among other virus or cell coded proteins.Another aspect of the invention features antibodies or antigen-bindingcomponents thereof that are immunospecific for epitopes presented by thepUL130-pUL128-containing complex, or that can otherwise prevent bindingof CMV to CD46 or another cell surface receptor through thepUL130-pUL128-containing complex. Another aspect of the inventionfeatures a method of diminishing CMV infection by administering one ormore of the aforementioned antibodies into a patient infected with CMV.Another aspect of the invention features an assay for antibodies thatare able to neutralize CMV infection of cells other than fibroblasts,e.g., epithelial and endothelial cells. Another aspect of the inventionsfeatures methods for identifying antiviral compounds that target theinteraction between the pUL130-pUL128-containing complex of CMV and itscellular receptor, CD46 or any other cell surface receptor with whichthe complex interacts.

Immunogenic Compositions and Vaccines, and Methods of Use

One aspect of the invention features an immunogenic compositioncomprising a pharmaceutically acceptable carrier and a complex ofcytomegalovirus (CMV) proteins comprising pUL128 or pUL130 and at leastone other virus or cellular constituent of a virion complex thatincludes pUL128 or pUL130. In one embodiment, the virion complexcomprises pUL128 or pUL130 alone. In another embodiment, the virioncomplex comprises pUL128 and pUL130. In another embodiment, the virioncomplex comprises glycoproteins gL and/or gH, or both. In anotherembodiment, the virion complex comprises pUL128, pUL130, gL and gH. Inanother embodiment, the virion complex comprises or is associated withpUL131. In yet another embodiment, fragments of the above-mentionedproteins are utilized, either as individual polypeptides or as fusionproducts.

Any CMV whose genome comprises a UL131-128 locus that is functional, orthat can be made functional through genetic manipulation, is suitablefor use as a source of the aforementioned virion complex or componentsthereof. In one embodiment, the CMV is human CMV. In another embodiment,the CMV originates from another primate, including but not limited tochimpanzee (Davison, A J et al. 2003, J. Gen. Virol. 84: 17-28) andrhesus monkey (Hansen, S G et al. 2003, J. Virol. 77:6620-36; Rivailler,P et al. 2006, J. Virol. 80:4179-82). The immunogenic composition maycomprise a virion complex with components all from the same CMV (e.g.,all from human CMV), or the components may be selected from CMVs ofdifferent species (e.g., pUL128 from human CMV, pUL130 from chimpanzeeCMV, and other such combinations).

The CMV virion complex or components thereof may be prepared in avariety of ways, in accordance with methods well known in the art. Forinstance, they may be isolated from the surfaces of virus particles andutilized together or separated into various components. In certainembodiments, the complex is produced by expression of one or morepolynucleotides encoding the CMV proteins, fragments of these proteins,or fused molecules. In certain embodiments, in addition to CMV proteinsand/or CMV protein fragments, fused molecules can include non-CMVproteins, non-CMV protein fragments, and/or synthetic fragments ofamino-acid sequence, as would be appreciated by the skilled artisan.

Nucleic acid sequences encoding the pUL128 and pUL130 proteins are knownin the art, and are provided, in whole or in part, in public databasessuch as those at the National Center for Biotechnology Information(NCBI). By way of example, and not of limitation, UL128 sequences areprovided at GenBank Accession Nos. DQ208272-DQ208294, and UL130sequences are provided at GenBank Accession Nos. DQ208254-208270, andDQ011966-DQ011969. The open reading frame of human CMV UL128 is about506-526 nucleotides in length, and is preferably 516 nucleotides inlength. The open reading frame encodes a protein of about 162 to about182 amino acids in length. Preferred encoded sequences are 172 aminoacids in length. The open reading frame of human CMV UL130 is about635-655 nucleotides in length, and is preferably 645 nucleotides inlength. The open reading frame encodes a protein of about 205 to about225 amino acids in length. Preferred encoded sequences are 215 aminoacids in length.

At least six strains of human CMV have been cloned as infectiousbacterial artificial chromosomes (BAC) and sequenced (Murphy, E et al.2003, Proc. Natl. Acad. Sci. USA 100: 14976-14981. The BAC sequences areavailable at GenBank Accession Nos. AC146999 (laboratory strain AD169,from which the BADrUL131 variant described herein was made); AC 146851(laboratory strain Towne); AC 146904 (clinical isolate PH); AC146905(clinical-like isolate Toledo); AC146906 (clinical isolate TR); andAC146907 (clinical isolate FIX). At least two strains of human CMV havebeen sequenced without prior BAC cloning, and are available at GenBankAccession Nos. BK000394 (laboratory strain AD 169) and AY446894(clinical isolate Merlin). The entire genome of a chimpanzee CMV strainis available at GenBank Accession No. AF480884. Utilizing the teachingsof the present application, the skilled artisan would be able to use anyof the aforementioned sequences, or any other publicly available CMVsequence to prepare the virions, pUL130 and/or pUL128-containing virioncomplexes or components thereof described herein.

Another aspect of the invention features a subunit vaccine for treatingan individual against infection with CMV. The subunit vaccines comprisea pharmaceutically acceptable carrier and an immunogenic CMV protein orprotein complex, such as the virion complex described above, thatincludes pUL128 and/or pUL130 or fragments of pUL128 and/or pUL130. Inone embodiment, the CMV protein or protein complex comprises pUL28. Inanother embodiment, the CMV protein or protein complex comprises pUL130.In another embodiment, the CMV protein or protein complex comprisespUL130 and pUL128. In another embodiment, the CMV protein or proteincomplex comprises other virion complex components or virion-associatedproteins, such as one or more of gL, gH or pUL131. These proteins,protein fragments, fused proteins, fused protein fragments andcomponents may be prepared by methods well known in the art, as setforth above in the description of the immunogenic compositions. It willbe understood by the skilled artisan that an effective vaccine need notcomprise an entire pUL128/pUL130 virion complex as described above. Itneed only comprise elements of that complex effective to elicit animmune response in a recipient sufficient to protect the recipient fromCMV infection upon exposure to CMV.

Any CMV whose genome comprises a UL131-128 locus that is functional, orthat can be made functional through genetic manipulation, is suitablefor use as a source of the aforementioned components of the subunitvaccine. In one embodiment, the CMV is human CMV. In another embodiment,the CMV originates from another primate, including but not limited tochimpanzee (Davison, A J et al. 2003, J. Gen. Virol. 84: 17-28) andrhesus monkey (Hansen, S G et al. 2003, J. Virol. 77:6620-36; Rivailler,P et al. 2006, J. Virol. 80:4179-82). The vaccine may comprisecomponents all from the same CMV (e.g., all from human CMV), or thecomponents may be selected from CMVs of different species (e.g., pUL128from human CMV, pUL130 from chimpanzee CMV, and other suchcombinations).

The subunit vaccine can further comprise an adjuvant. Adjuvants can beany substance that enhances the immune response to the antigens in thevaccine. Non-limiting examples of adjuvants suitable for use in thepresent invention include Freund's adjuvant, incomplete Freund'sadjuvant, saponin, surfactants such as hexadecylamine, octadecylamine,lysolecithin, demethyldioactadecyl ammonium bromide,N,N-dioctadecyl-N′—N-bis (2-hydroxyethylpropane diamine),methoxyhexa-decyl-glycerol, pluronic polyols, polyanions such as pyran,diethylaminoethyl (DEAE) dextran, dextran sulfate, polybrene, poly IC,polyacrylic acid, carbopol, ethylene maleic acid, aluminum hydroxide,and aluminum phosphate peptides, oil or hydrocarbon emulsions, and thelike.

The protein or protein complex including pUL128 and/or pUL130 orfragments of pUL128 and/or pUL130 can be coupled to a carrier protein.It is within the skill in the art to select suitable carrier proteins tocouple to the CMV protein complex. Non-limiting examples of suitablecarrier proteins include albumin, ovalbumin, Pseudomonas exotoxin,tetanus toxin, ricin toxin, diphtheria toxin, cholera toxin, heat labileenterotoxin, keyhole lympet hemocyanin, epidermal growth factor,fibroblast growth factor, transferring, platelet-derived growth factor,poly-L-lysine, poly-L-glutamine, mannose-6-phosphate, as well as variouscell surface and membrane proteins, and the like.

In some embodiments, the protein or protein complex including pUL128and/or pUL130 or fragments of pUL128 and/or pUL130 is expressed on thesurface of an attenuated CMV virus particle. Methods to attenuateviruses are known in the art. For example, serial passage in fibroblastscan be used to attenuate CMV. Repeated passaging of virally-infectedhost cells is carried out in vitro until sufficient attenuation of thevirus is achieved. Passaging may be conducted under specificenvironmental conditions, such as modulated temperature, pH, humidity,in order to select for viruses with reduced infectivity orpathogenicity. Mutagenesis can also be employed. For example, CMVvirions can be exposed to ultraviolet or ionizing radiation or chemicalmutagens, according to techniques known in the art. Recombinanttechniques can also be used to produce attenuated CMV virions. Forexample, site-directed mutagenesis, gene replacement, or gene knockouttechniques can be used to derive virus strains with attenuatedinfectivity or pathogenicity. Preferably, attenuated CMV exhibit adiminished capacity for infectivity, and/or pathogenicity, yet remaincapable of inducing an immune response that treats or protects the hostagainst CMV infection. Examples of attenuated CMV strains include, butare not limited to, laboratory strains, such as AD169 and Towne, whichreplicate almost exclusively in fibroblasts. Such attenuated strains,engineered to produce a surface protein or protein complex includingpUL128 and/or pUL130 or fragments of pUL128 and/or pUL130, could begrown in fibroblasts or other cell types, e.g., epithelial cells, foruse as a vaccine.

In some embodiments, the pUL128 and/or pUL130 protein or proteincomplex, and derivatives thereof described herein may be administered asa component of a more complex vaccine that includes additional CMV geneproducts. These additional CMV gene products can be complete proteinsand/or fragments of proteins. In one embodiment, immunogenic fragmentsof pUL128 and/or pUL130 may presented alone or may be combined into onepolypeptide chain that includes immunogenic fragments of additionalcomponents of the pUL128-pUL130 virion complex, e.g., gH, and/oradditional components of the virion, e.g., gB. Such polypeptidescomprised of multiple immunogenic fragments may also contain non-CMVand/or synthetic amino acid sequences.

In some embodiments, the pUL128 and/or pUL130 protein or proteincomplex, and derivatives thereof described herein, can be administeredas components of a vaccine vector. Vaccine vectors include modifiedviruses, bacteria and other microbes. For example, an adenovirusderivative can be produced that lacks one or more adenovirus genes orgene fragments and contains in its/their place nucleic acid encoding thepUL128 and/or pUL130 protein or protein complex, and derivativesthereof.

Vaccines can be formulated in aqueous solutions such as water oralcohol, or in physiologically compatible buffers such as Hanks'solution, Ringer's solution, or physiological saline buffer, includingPBS. Vaccine formulations can also be prepared as solid formpreparations which are intended to be converted, shortly before use, toliquid form preparations suitable for administration to a subject, forexample, by constitution with a suitable vehicle, such as sterile water,saline solution, or alcohol, before use.

The vaccine compositions can also be formulated using sustained releasevehicles or depot preparations. Such long acting formulations may beadministered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thevaccines may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt. Liposomes and emulsions can be used as deliveryvehicles suitable for use with hydrophobic formulations.Sustained-release vehicles may, depending on their chemical nature,release the antigens over a range of several hours to several days toseveral weeks to several months.

The vaccine compositions may further include one or more antioxidants.Exemplary reducing agents include mercaptopropionyl glycine,N-acetylcysteine, 3-mercaptoethylamine, glutathione, ascorbic acid andits salts, sulfite, or sodium metabisulfite, or similar species. Inaddition, antioxidants can also include natural antioxidants such asvitamin E, C, leutein, xanthine, beta carotene and minerals such as zincand selenium.

Vaccine compositions may further incorporate additional substances tofunction as stabilizing agents, preservatives, buffers, wetting agents,emulsifying agents, dispersing agents, and monosaccharides,polysaccharides, and salts for varying the osmotic balance. The vaccinescan further comprise immunostimulatory molecules to enhance vaccineefficacy. Such molecules can potentiate the immune response, can induceinflammation, and can be any lymphokine or cytokine. Nonlimitingexamples of cytokines include interleukin (IL)-1, IL-2, IL-3, IL-4,IL-12, IL-13, granulocyte-macrophage colony stimulating factor (GMCSF),macrophage inflammatory factor, and the like.

Subunit vaccines can be formulated for and administered by infusion orinjection (intravenously, intraarterially, intramuscularly,intracutaneously, subcutaneously, intrathecally, intraduodenally,intraperitoneally, and the like). The vaccines can also be administeredintranasally, vaginally, rectally, orally, topically, buccally,transmucosally, or transdermally.

An effective antigen dosage to treat against CMV infection can bedetermined empirically, by means that are well established in the art.The effective dose of the vaccine may depend on any number of variables,including without limitation, the size, height, weight, age, sex,overall health of the subject, the type of formulation, the mode ormanner or administration, whether the virus is active or latent, whetherthe patient is suffering from secondary infections, or other relatedconditions.

Vaccine regimens can also be based on the above-described factors.Vaccination can occur at any time during the lifetime of the subject,including development of the fetus through adulthood. Supplementaladministrations, or boosters, may be required for full protection. Todetermine whether adequate immune protection has been achieved,seroconversion and antibody titers can be monitored in the patientfollowing vaccination.

The invention also features nucleic acid vaccines for treating againstCMV infection. In general, nucleic acid vaccines, also referred to asgenetic vaccines, utilize DNA or RNA encoding a antigen of interest, andrely on host expression of the genes to stimulate an immune response tothe encoded polypeptide (Leitner W W et al. (2000) Vaccine 18:765-77).The nucleic acid vaccines of the present invention comprise apharmaceutically acceptable carrier and at least one nucleic acidmolecule encoding an immunogenic CMV protein or protein complexincluding pUL128 and/or pUL130 and possibly other virus and/or cellcoded proteins, or fragments thereof that elicit an immune response. Thenucleic acid molecule is expressed in host cells of the vaccinerecipient, and the expression product induces an immune response to theCMV protein complex including pUL128 and/or pUL130. The immune responsetreats against CMV infection. Nucleic acid sequences encoding variousCMVs and components thereof are known in the art, as described in detailabove.

Nucleic acid vaccines can be formulated to target specific cells or celltypes. For example, it may be preferred to target antigen presentingcells such as dendritic cells, monocytes, macrophages, B cells, and thelike.

Antibodies and Methods of Use

Also featured in the present invention are antibodies that specificallyrecognize epitopes within pUL128 or pUL130 or any constituent of thecomplex that includes pUL128 and pUL130 in the virus particle. These mayinclude one or more of glycoprotein gH, glycoprotein gL or pUL131. Inpreferred embodiments, the antibodies inhibit CMV infection of a cell byblocking the ability of the virus to bind to receptors on the cellsurface. Such antibodies are sometimes referred to herein asneutralizing antibodies, in accordance with the art-recognizeddefinition.

Any antibody that specifically binds to pUL128 or pUL130 or to anyconstituent of the protein complex that includes these proteins invirions, can be used in the present invention. Any antibody thatspecifically binds to CD46 can also be used in the present invention.Monoclonal (single antibodies or mixtures of antibodies) and/orpolyclonal antibodies can be used, from whatever source produced arepreferred, although recombinant antibodies such as single chainantibodies and phage-displayed antibodies, and antigen binding fragmentsof antibodies such as the Fab or Fv can also be used. Antibodies thatrecognize pUL128, pUL130, any constituents of the CMV protein complexincluding pUL 128 and pUL130, and CD46, can be used in the invention. Inone embodiment, the antibodies recognize epitopes of pUL128, pUL130, orother components of the virion complex as they are presented in thecomplex, but do not recognize pUL128 or pUL130, or other components ofthe virion complex in solution or otherwise apart from the virioncomplex. In another embodiment, the antibodies recognize epitopes ofpUL128 or pUL130, or other components of the virion complex in solutionor otherwise apart from the virion complex. Regardless of how theantibodies are produced, preferred embodiments of the invention utilizeor are directed to neutralizing antibodies. Indeed, another aspect ofthe invention, set forth below, features an assay to identify desiredneutralizing antibodies. Methods for raising and purifying antibodiesare well known in the art. In addition, monoclonal antibodies can beprepared by any number of techniques that are known in the art,including the technique originally developed by Kohler and Milstein(1975) Nature 256:495-497.

Antibodies suitable for use in the methods of the invention include, forexample, fully human antibodies, single chain antibodies, human antibodyhomologs, humanized antibody homologs, chimeric antibodies, chimericantibody homologs, and monomers or dimers of antibody heavy or lightchains or mixtures thereof. The antibodies of the invention can beintact immunoglobulins of any isotype, including types IgA, IgG, IgE,IgD, IgM (as well as all subtypes and idiotypes thereof). The lightchains of the immunoglobulin may be kappa or lambda. The antibodies canbe portions of intact antibodies that retain antigen-bindingspecificity, for example, Fab fragments, Fab′ fragments, F(ab′)₂fragments, F(v) fragments, heavy chain monomers or dimers, light chainmonomers or dimers, dimers consisting of one heavy and one light chain,and the like. Recombinant antibodies, including single chain antibodiesand phage-displayed antibodies, diabodies, as well as individualantibody light chains, individual antibody heavy chains, chimericfusions between antibody chains and other molecules, heavy chainmonomers or dimers, light chain monomers or dimers, dimers consisting ofone heavy and one light chain, and the like, can also be used.

The antibodies of the invention can be modified, e.g., by the covalentattachment of any type of molecule to the antibody such that covalentattachment does not prevent the antibody from binding to its epitope.Examples of suitable derivatives include, but are not limited toglycosylated antibodies and fragments, acetylated antibodies andfragments, pegylated antibodies and fragments, phosphorylated antibodiesand fragments, and amidated antibodies and fragments. In onenon-limiting example, functional attributes of the antibodies orfragments many be modulated by their production in yeast expressinghuman pathways that mediate the generation of antibodies with specificglycosylated structures (Li, H. et al., 2006, Nat. Biotech. 24:210-5).The antibodies and derivatives thereof of the invention may themselvesbe derivatized by known protecting/blocking groups,proteolytic-cleavage, linkage to a cellular ligand or other proteins,and the like. Further, the antibodies and derivatives thereof of theinvention may contain one or more non-classical amino acids.

The antibodies of the invention can be variants having single ormultiple amino acid substitutions, deletions, additions, or replacementsthat retain the biological properties (e.g., internalization, bindingaffinity or avidity, or immune effector activity) of the antibodies ofthe invention. The skilled artisan can produce variants having single ormultiple amino acid substitutions, deletions, additions or replacements.These variants can include, among other things (a) variants in which oneor more amino acid residues are substituted with conservative ornonconservative amino acids, (b) variants in which one or more aminoacids are added to or deleted from the polypeptide, (c) variants inwhich one or more amino acids include a substituent group, and (d)variants in which the polypeptide is fused with another peptide orpolypeptide such as a fusion partner, a protein tag or other chemicalmoiety, that may confer useful properties to the polypeptide, such as,for example, an epitope for an antibody, a polyhistidine sequence, abiotin moiety and the like.

Antibodies can be labeled/conjugated to various moieties, includingdetectable moieties and drugs/toxins. Drug/toxin moieties include, forexample, bacterial toxins, viral toxins, organic chemicals, inorganicchemicals, radioisotopes, and the like. Antibodies can be labeled foruse in biological assays (e.g., radioisotope labels, fluorescent labels)to aid in detection of the antibody. Antibodies may also be conjugatedwith toxins to provide an immunotoxin (see, Kreitman R J (1998). Adv.Drug Del. Rev., 31:53). Detectable moieties contemplated for use in theinvention include, but are not limited to, radioisotopes, fluorescentdyes such as fluorescein, phyocoerythrin, Cy-3, Cy5, allophycocyanin,DAPI, Texas red, rhodamine, Oregon green, lucifer yellow, and the like,green fluorescent protein, red fluorescent protein, cyan fluorescentprotein, yellow fluorescent protein, Cerianthus orange fluorescentprotein, alkaline phosphatase, β-lactamase, chloramphenicolacetyltransferase, adenosine deaminase, aminoglycosidephosphotransferase (neo^(r), G418^(r)) dihydrofolate reductase,hygromycin-B-phosphotransferase, thymidine kinase, lacZ (encodingα-galactosidase), and xanthine guanine phosphoribosyltransferase,β-glucuronidase, placental alkaline phosphatase, secreted embryonicalkaline phosphatase, or firefly or bacterial luciferase. Enzyme tagsare used with their cognate substrate. As with other standard proceduresassociated with the practice of the invention, skilled artisans will beaware of additional labels that can be used. In some embodiments, theantibody is conjugated to biotin, and subsequently contacted with avidinor strepatvidin having a detectable moiety tag.

As described in Example 2, murine monoclonal antibodies (mAbs) specificfor pUL130 (3E3 and 3C5) and pUL128 (4B10), as well as rabbitanti-pUL128 polyclonal antibody (R551A), were generated by using GSTfusion proteins as immunogens. Antiserum containing polyclonal antibodyR551A, as well as hybridoma cell lines producing mAb 3E3 and 4B 10,respectively, were deposited with the patent depository of the AmericanType Culture Collection (ATCC) on Jun. 5, 2007 and given the followingATCC designations: ATCC Accession No. PTA-8474 for antiserum R551A; ATCCAccession No. PTA-8472 for the hybridoma producing mAB 3E3; and ATCCAccession No. PTA-8473 for mAb 4B10. These antibodies, or fragmentsthereof, may be used to practice the many aspects of the inventiondescribed herein.

Furthermore, it is well within the purview of the skilled artisan toobtain or design variants and/or derivatives of the depositedantibodies, e.g., through competitive binding assays or through geneticmanipulation, which are able to bind pUL128 or pUL130, or complexescontaining pUL128 or pUL130. Methods for determining antibodyspecificity and affinity by competitive inhibition can be found inHarlow, et al., Antibodies: A Laboratory Manual, 1988; supra; Colliganet al., eds., 1992, 1993, Current Protocols in Immunology, GreenePublishing Assoc. and Wiley Interscience, N.Y., and Muller, 1983, Meth.Enzymol. 92:589-601.

For instance, antibodies that compete for binding to an epitope onpUL128 or pUL130 recognized by the deposited mAbs, or antibodies thatbind to the same epitope(s) as the deposited mAbs, are suitable for usein the present invention. In addition, a neutralizing binding partner ofa CMV virion complex comprising pUL128 or pUL130, which comprises one ormore complementarity determining regions (CDRs) having 70% or greateridentity to a CDR present in the deposited mAbs, is also contemplatedfor use in the present invention. Preferably, the neutralizing bindingpartner has two or three CDRs with the requisite degree of identity.Preferably, the percent similarity in one or more of the CDRs is 75%,80%, 85%, 90% 95% or more identical to one or more of the CDRs of thedeposited mAbs. Methods of determining the sequence of antibody CDRs arewell known to the skilled artisan, as are methods of designing suchneutralizing binding partners.

Another aspect of the invention features a method of diminishing CMVinfection by introducing antibodies that block binding of CMV throughits pUL128-pUL130-containing complex to a host cell, e.g., through acellular receptor, such as CD46, which is a target of thepUL128-pUL130-containing complex, in a CMV infected subject. In variousembodiments, the antibodies are immunologically specific for one or moreepitopes of constituents of the pUL130-pUL128-containing complex aspresented when the proteins are associated as a complex on the surfaceof a virus particle or on a carrier protein or free in solution. In oneembodiment, the antibodies are immunospecific for one or more epitopesof pUL130, while in another embodiment they are immunospecific for oneor more epitopes of pUL128.

Assays

The invention also features methods for screening compounds that inhibitthe interaction between the CMV pUL130-pUL128-containing surface complexand cellular receptors or mediators of viral entry on the surfaces ofhost cells. In one embodiment, the screening assays involve contacting aCMV particle with a test compound, and contacting the testcompound-treated CMV particle with CD46 or a cell or membrane fragmentcomprising CD46, and determining whether the test compound inhibits theCMV particle binding to CD46. Candidate compounds to be tested by themethods of the present invention include purified molecules,substantially purified molecules, molecules that are one or morecomponents of a mixture of compounds, or a mixture of a compound withany other material. Test compounds can be organic or inorganicchemicals, or biomolecules, and all fragments, analogs, homologs,conjugates, and derivatives thereof. Test compounds can be of natural orsynthetic origin, and can be isolated or purified from their naturallyoccurring sources, or can be synthesized de novo. Test compounds can bedefined in terms of structure or composition, or can be undefined. Thecompound can be an isolated product of unknown structure, a mixture ofseveral known products, or an undefined composition comprising one ormore compounds. Examples of undefined compositions include cell andtissue extracts, growth medium in which prokaryotic, eukaryotic, andarchaebacterial cells have been cultured, fermentation broths, proteinexpression libraries, and the like. In one embodiment of this invention,the test compound could be small peptides comprising amino acidsequences corresponding to constituents of the pUL128-pUL130-containingcomplex. The peptides can contain naturally occurring amino acids,chemically modified amino acids and/or synthetic derivatives of aminoacids. In a preferred embodiment of this invention, the peptides can be8-20 amino acid units in length.

In one embodiment, the screening assays comprise contacting a CMVprotein or protein complex including pUL128 and/or pUL130 or a fragmentof pUL128 and/or pUL130, with a test compound, and contacting the testcompound-treated protein or protein complex with a host cell, cellmembrane or membrane fraction comprising one or more cellular receptorsto which the pUL128 and/or pUL130 complex binds, and determining whetherthe test compound inhibits the protein or protein complex binding to thecell or cell membrane. In another embodiment, the screening assayscomprise first contacting the cell or membrane fraction with a testcompound, and then contacting the cell or membrane fraction, with a CMVprotein or protein complex including pUL128 and/or pUL130, anddetermining whether the test compound inhibits binding of the CMVprotein or protein complex including LTL 128 and UL130 to the cell ormembrane fraction.

In some embodiments, the host cell or membrane fraction comprising areceptor such as CD46, is immobilized on a solid support. In otherpreferred embodiments, the CMV protein or protein complex includingpUL128 and/or pUL30 is immobilized on a solid support. Examples ofsuitable solid supports include, but are not limited to, glass, plastic,metal, latex, rubber, ceramic, polymers such as polypropylene,polyvinylidene difluoride, polyethylene, polystyrene, andpolyacrylamide, dextran, cellulose, nitrocellulose, pvdf, nylon,amylase, and the like. A solid support can be flat, concave, or convex,spherical, cylindrical, and the like, and can be particles, beads,membranes, strands, precipitates, gels, sheets, containers, wells,capillaries, films, plates, slides, and the like. The solid support canbe magnetic, or a column.

For the screening assays, host cells and cell membrane fractions, CMV,or the CMV protein or protein complex including UL128 and/or UTL130 canbe obtained from any source suitable in the art and prepared accordingto art-recognized methods. For instance, the BADrUL131 strainexemplified herein produces virions comprising gH-gL-pUL130-pUL128complex. As mentioned, CD46 can be purified or bound to a cell membraneor membrane fragment. Purified CD46, or subunits thereof, can besynthesized de novo, or obtained from any mammalian cell that naturallyexpresses CD46. Host cells can include endothelial cells and epithelialcells. Constituents of the pUL130-pUL128 complex can be purified fromthe CMV virion. Purified virus proteins or a fused pUL130-128 complexcan also be produced from recombinant expression systems, such asbacterial, yeast, insect cell systems, and the like. Techniques ofrecombinant cloning and protein expression and purification are wellestablished in the art.

In another embodiment, candidate antiviral compounds shown to affectbinding of the pUL130-128-containing complex to a host cell, cellmembrane or cellular receptor can be further evaluated for their abilityto reduce infectivity. This can be accomplished in a variety of waysknown in the art, for example, by plaque reduction assays as describedin the Examples herein or by monitoring the expression of a de novosynthesized protein encoded from the viral genome. Candidate antiviralcompounds can also be tested in assays designed to measure membranefusion, preferably at both the level of virus-cell and cell-to-cellspread of infection. Induction of infection and cell-to-cell spread canbe monitored in many ways, including observation of cytopathic effect ormonitoring the spread of virus proteins or marker proteins incorporatedinto the viral genome by immunofluorescence.

Another aspect of the invention features a cell culture assay by whichto identify and quantify the CMV neutralizing activity that has beengenerated after immunization with vaccine candidates. Thisneutralization assay can be used to monitor potential efficacy of avaccine in preclinical animal models; this is useful because human CMVdoes not infect non-human animal models and it is not feasible toperform human CMV immunization-challenge experiments in animal modelsprior to human trials. This assay also can be used to provide aquantitative measure of a critical aspect of vaccine function duringclinical trials, i.e., the generation of neutralizing antibodies. Animportant underpinning of this aspect of the invention is the use ofcell types than can be infected only by human CMV containing afunctional pUL128-pUL130-containing complex, e.g., epithelial cells.Normal, unmodified human fibroblasts, a cell commonly used for analysisof human CMV infectivity would be ineffective in this assay becausepUL128-pUL130-containing virions do not infect fibroblasts well (seeExamples herein). Those of ordinary skill in the art can implement anassay for neutralization of human CMV infectivity of the appropriatecells. As a non-limiting example, a defined amount of infectious humanCMV which contains on its surface the pUL128-pUL130-containing complexis mixed with dilutions of animal or human serum or antibodies derivedfrom serum, and then the mixture is added to monolayers of epithelialcells, such as ARPE-19 cells, and the entry of virus into the cells ismonitored. Entry can be monitored by many methods, including the de novoexpression of a viral gene product or marker gene incorporated into theviral genome or by plaque-reduction assay as described in the Examplesherein. By comparing the number of cells infected by untreated virus tothe number of cells infected by virus after exposure to serum sample, itis possible to calculate the reduction in infectivity, orneutralization, caused by antibodies in the samples.

The following examples are provided to describe the invention in moredetail. They are intended to illustrate, not to limit, the invention.

Example 1 Human Cytommalovirus UL131 ORF is Required for Epithelial CellTropism

This example describes a systematic investigation of human CMV infectionof epithelial cells, using a panel of cultured cells that originatedfrom different organs and tissues. It was determined that culturedepithelial cells can be efficiently infected by human CMV strains with awild-type UL131-UL128 locus. The AD169 laboratory strain can efficientlyinfect both epithelial and endothelial cells when the mutation in itsUL131 ORF is repaired.

Materials and Methods

Cells and Viruses.

Primary human foreskin fibroblasts (HFFs) at passage 10-15 weremaintained in Dulbecco's minimal essential medium (DMEM) supplementedwith 10% new born calf serum. Two types of endothelial cells,immortalized human umbilical vein endothelial cells (HUVECs) and lungmacovascular endothelial cells (LMVECs), were obtained from privatesources but are otherwise available or can be obtained. The endothelialcells were cultured in EGM-2 medium (Combrex, East Rutherford, N.J.).HeLa cells, primary human MRC-5 embryonic lung fibroblasts and ARPE-19retinal pigmented epithelial cells were purchased from ATCC. Both MRC-5cells and ARPE-19 cells were used at passage 24-30. The MRC-5 cells werecultured in DMEM medium supplemented with 10% fetal bovine medium, andARPE-19 cells were propagated in DMEM/Ham's F12 (1:1) medium containing10% of fetal bovine serum. SW480, HCT116, 111299, MCF-7, SK-N-SH,SK-N-AS, IMR-32 cells were propagated in DMEM supplemented with 10%fetal bovine serum.

A derivative of the AD169 strain of human CMV (Yu, D et al., 2002, JVirol 76: 2316-2328) with a GFP marker (BADwt) was used as the parentalAD169 virus. In this variant, the UL21.5 region of the virus wasreplaced with a marker cassette containing the GFP coding region undercontrol of a SV40 promoter followed by an internal ribosome entry siteand a puromycin resistance gene (Wang, D et al., 2004, Proc. Natl. Acad.Sci. USA 101: 16642-166427). To repair the AD 169 UL131 coding sequence,linear recombination (Borst, E M S et al., 2001, J. Virol. 75:1450-1458)was employed to substitute the human CMV sequence from base pair 176685to 176794 (sequence numbers according to Chee, M S et al., 1990, Curr.Top. Microbiol. Immunol. 154: 125-169) with a marker cassette containingkanamycin-resistance and LacZ genes in an infectious BAC clone carryingthe BADwt genome. This clone was then transformed into E. coli GS500,and allelic exchange was performed with a pGS284 (Smith, G A and L WEnquist, 1999, J. Virol. 73: 6405-6414) derivative carrying the UL131ORF and flanking sequences from the human CMV clinical TR strain (Smith,I L et al., 1998, Arch. Ophthalmol. 116: 178-185). To determine therecombination sites between the BAC clone and the shuttle plasmid, theUL131-UL128 locus were sequenced. Two repaired UL131 BAC clones withdifferent N terminal protein sequences were generated by allelicexchange (FIG. 1). The viruses recovered from the repaired BAC cloneswere designated BADrUL131-Y4 and BADrUL131-C4.

Wild-type virus stocks were prepared from BAC clones of human CMVlaboratory strains and clinical isolates: Towne (BTNwt) (Marchini, A etal., 2001, J. Virol. 75: 1870-1878), Toledo (BLTwt) (Murphy, E et al.,2003, supra), VR1814-FIX (BFXwt) (Hahn G et al., 2004, supra) and TR(BTRwt) ((Murphy, E et al., 2003, supra). BTNwt lacks US1-US12, BLTwtlacks US2-US11, BFXwt lacks IRS 1-US6, and BTRwt lacks US2-US5 ((Murphy,E et al., 2003, supra) as a consequence of BAC cloning. Viruses werereconstituted by electroporation of BAC DNA into HFFs, together with aplasmid expressing the viral UL82-coded pp71 protein, which enhances theinfectivity of human CMV DNA. Virus stocks were prepared in HFF cells,and the first passage of each virus preparation was used for celltropism studies.

Assays for virus infection and replication. To monitor the efficiency ofinfection, epithelial cells, endothelial cells or fibroblasts weretransferred into 12-well plates and incubated overnight to producesubconfluent cultures. Monolayers were washed once with serum-freeRPMI-1640 and infected with virus diluted in the same medium at amultiplicity of 1 pfu/cell. During the adsorption period, the plateswere first subjected to centrifugation at 25° C. for 30 min at 1,000×g,and then incubated at 37° C. for 1 h. The inoculum was removed and freshmedium containing the serum appropriate to the cell type was added. At48 h after infection, cultures were fixed in 2% paraformaldehyde andpermeabilized with 0.1% Triton-X 100, and infected cells were identifiedby GFP expression and IE1 by immunofluorescence using monoclonalantibody 1 B 10 and Alexa 546-conjugated secondary antibody. The nucleiof cells were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Totalcell numbers were determined by the number of DAPI-stained nuclei, andefficiencies of infection were calculated as the percentage of GFP andIE1 expressing cells in the total cell populations.

For analysis of virus growth kinetics, cells were infected at amultiplicity of 0.01 or 1 pfu/cell with BADwt (laboratory strain parent)or BADrUL131 viruses (repaired UL 131 ORF), with the exception of HeLacells, which were only infected at a multiplicity of infection of 1pfu/cell because HeLa cell cultures could not be maintained for theextended period of time needed for analysis of a low multiplicityinfection. At various times after infection, cell-free virus wascollected by harvesting medium from infected cultures andcell-associated virus was collected by three freeze-thaw cycles ofinfected cells in medium three times. Virus titers were determined byTCID₅₀ assay on MRC-5 cells. In contrast to parental AD169, theUL131-repaired viruses induced syncytia and exhibited reduced plaqueforming efficiencies. Therefore, to compare the growth of the mutantsand their wild-type parent, we relied on GFP gene expression rather thancytopathic effect to identify the infected wells in our TCID₅₀ assays.The use of the GFP marker carried on the viruses significantly increasedthe sensitivity and accuracy of the assays.

Results

Construction and Characterization of an AD169 Variant with a RepairedUL131 ORF.

Two spliced transcripts with a common polyA addition site were shown tobe generated by the UL131-UL128 locus (Akter P C et al., 2003, supra;Hahn G et al., 2004, supra), but their 5′ ends were not localized. Tofurther characterize the transcripts produced by this locus, we mappedthe two start sites (FIG. 1A) by using 5′-RACE analysis, and weconfirmed the result using RNase protection assays (data not shown). The5′ ends are located at sequence positions 176835, just upstream ofUL131, and 176207, within UL130 (numbering according to Chee, M S etal., 1990, supra). The mRNA with the 5′ end upstream of the UL131 codingregion has the potential to encode polypeptides encoded by all threeORFs, whereas the RNA whose 5′ end maps within UL130 has the potentialto encode a portion of UL130 and UL128. This 5′ mapping confirms thatthe one base-pair insertion present in the UL131 ORF of AD169 (FIG. 1A)is, indeed, present within an mRNA that has been mapped to this locus.

The frame-shifted UL131 ORF in AD169 was repaired by constructingderivatives of the laboratory strain (BADrUL131) in which the mutatedUL131 ORF was replaced with a wild-type UL131 ORF derived from the humanCMV TR clinical strain (FIG. 1A). Two different viruses with a repairedUL131 ORF were generated, due to recombination events at two differentsites between the BAC clone and the shuttle plasmid. The sequences ofthe UL131 ORFs from the newly generated viruses were compared with UL131ORFs from Towne, Toledo, FIX and TR strains (FIG. 1B). Except foramino-acid positions 2 and 4, the sequences of UL131 ORFs from differentclinical human CMV strains are identical. The UL131 sequence ofBADrUL131-Y4 derives entirely from the TR strain, while the BADrUL131-C4sequence is identical to that of FIX and Toledo strains (FIG. 1B).

It was previously shown (Hahn et al., 2004, supra) that a UL131 deletionmutant of the clinical isolate FIX, a BAC-cloned derivative of VR1814(Grazia Revello, M et al., 2001, J. Gen. Virol. 82:1429-1438), is nolonger able to replicate in endothelial cells. To confirm thatendothelial cell tropism was restored by repairing the UL131 gene in AD169, two endothelial cell lines, HUVECs and LMVECs, were infected atmultiplicity of 1 pfu/cell, and the cultures were assayed 48 h later forexpression of the GFP marker carried by the viruses. Very few (<1%)GFP-expressing HUVECs were seen in BADwt infected cultures; in contrast,GFP could be detected in almost all the HUVECs infected by bothBADrUL131 viruses, demonstrating that the repaired AD169 variants hadreacquired the ability to infect endothelial cells. LMVECs were moreefficiently infected by BADwt (12%), but the efficiency of infection wasconsiderably greater for BADrUL131-Y4 (96%) and BADrUL131-C4 (98%). Incontrast to the UL131-deficient parent, the repaired BADrUL131 virusesinduced syncytia in infected cultures of both fibroblasts andendothelial cells.

To evaluate the growth characteristics of the repaired viruses, MRC-5fibroblasts and HUVECs were infected with BADwt or BADrUL131 variants,and the production of cell-free and cell-associated virus in single stepgrowth or multi-step growth analysis was assayed. The repaired virusesgrew more poorly than their AD 169 parent in fibroblasts, generating a10 to 100-fold reduced yield of both cell-free and cell-associatedvirus. The reduced yields correlated with extensive cell-cell fusioninduced by the repaired viruses, although it is not clear that cellfusion compromises the efficiency of virus replication in fibroblasts.In contrast to fibroblasts, the repaired viruses grew more efficientlyin endothelial cells than their laboratory-strain parent, producingabout 20-fold more cell-free and 300-fold more cell-associated virus.

Collectively, these data demonstrate that repair of the mutated UL131gene in AD169 compromises virus replication in fibroblasts andfacilitates growth in endothelial cells.

UL128-131 Locus-Dependent Infection of Epithelial Cells.

The importance of UL131 for human CMV replication in epithelial cellswas evaluated by examining the susceptibility of a panel of epithelialcell lines to BADwt and BADrUL131-Y4. Susceptibility to infection wasassayed by monitoring expression of the UL123-coded IE1 protein andexpression of the GFP marker genes carried by the two viruses. Theepithelial cell lines originated from a variety of tissues: retina(ARPE-19), cervix (HeLa), colon (SW480 and HCT116), lung (H1299) andbreast (MCF-7). It was found that HeLa and MCF-7 cells could beefficiently infected by BADrUL131, and the infection was highlydependent on UL 131 since a very low incidence f infection was seen forthe AD 169 parent, BADwt. Cell-cell fusion in the MCF-7 epithelialcells, but not in HeLa cell cultures, was seen after infection withBADrUL131-Y4. This also demonstrated that GFP expression can lead to anunderestimate of the percentage of cells that are infected, because thehuman CMV UL123-coded IE1 protein was detected in a greater proportionof cells. Presumably this reflects differences in the activity of theSV40 early promoter, which controls the expression of GFP in theseviruses, relative to the human CMV immediate-early promoter.

To quantify the relative susceptibilities of the epithelial cell lines,a calculation was made of the percentage of cells expressing GFP fromthe viral genome in different epithelial cell populations afterinfection at identical input multiplicities with the parental virus orrepaired virus. A repaired UL131 ORF was absolutely required to infectARPE-19, HeLa, SW480, HCT116 and MCF-7 epithelial cells. The repairedORF was not essential, but dramatically enhanced the efficiency ofinfection, in H1299 cells. Fibroblasts and endothelial cells wereincluded as controls. Although no detectable differences insusceptibility to infection with BADwt versus BADrUL131-Y4 were evidentin MRC-5 or HFF fibroblasts, GFP expression in HUVEC and LMVECendothelial cells was dramatically enhanced. Finally, severalneuron-derived cell lines were tested. The SK-N-AS, SK-N-SH and IMR-32neuroblastoma cell lines showed variable susceptibilities to infection,but GFP expression was not UL 131 gene dependent.

The production of infectious progeny by the BADrUL131 repaired viruseswas examined in ARPE-19 cells. The epithelial cells were infected at amultiplicity of 0.01 or 1 pfu/cell and the production of cell-associatedand cell-free virus was monitored by TCID₅₀ assay on fibroblasts. Inepithelial cells, BADrUL131-Y4 and BADrUL131-C4 produced a ≥10-foldgreater yield than in fibroblasts and an ˜100-fold greater yield than inendothelial cells. Further, BADrUL131 induced extensive cell-cell fusionin epithelial cell cultures, as it did in fibroblasts and endothelialcells.

The replication of the two BADrUL131 repaired viruses also was monitoredin HeLa cells, an epithelial tumor cell line. Infection of HeLa cellsgenerated a yield of cell-free virus that was reduced by a factor of˜1000 in comparison to the yield from APRE-19 cells. It is noteworthythat 98% of HeLa cells and 77% of ARPE-19 cells expressed the GFP markerafter infection at a multiplicity, determined by TCID₅₀ assay onfibroblasts, of 1 pfu/cell with BADrUL131-Y4. In spite of the higherefficiency with which the marker was expressed in HeLa cells, the yieldof infectious progeny was greater in ARPE-19 cells. Apparently, there isa block to efficient replication of human CMV in HeLa cells, relative tothe efficiency of replication in ARPE-19 cells, which occurs after thegenome reaches the nucleus and expresses the marker gene. In contrast toARPE-19 cells, HeLa cells fail to undergo fusion after infection withBADrUL131 viruses, although significant cytopathic effects withcharacteristic cell rounding can be detected as early as 24 h afterinfection. This might reflect a failure of the virus to efficientlyadvance to the late stage of the replication cycle. This would inhibitexpression of the UL131 ORF, which is expressed during the late phase.

A mutation in UL131 is responsible for the inability of AD 169 toefficiently replicate in epithelial cells. To further elucidate the roleof the UL131-128 locus in human CMV epithelial cell tropism, assays forinfection by four additional human CMV strains, Towne, Toledo, FIX andTR, were performed. UL130 is mutated in Towne and UL128 is disrupted inToledo, whereas FIX and TR contain wild-type UL128, 130 and 131 ORFs.All of the virus strains infected the MRC-5 fibroblasts and expressedtheir IE1 protein equally well, but only TR and FIX efficiently infectedHUVEC endothelial or ARPE-19 and HeLa epithelial cells. This resultdemonstrates that the Towne and Toledo strains do not efficiently enterepithelial cells and express their IE1 genes, and raises the possibilitythat the defect might result from mutations in the UL130 and UL128 ORFs,as has been shown for replication in endothelial cells (Hahn et al.,2004, supra).

Finally, the yields of infectious progeny were determined afterinfection of ARPE-19 epithelial cells with the set of human CMV strains.The repaired AD169 strain, BADrUL131-Y4, produced the highest yield inepithelial cells (1×10⁷ pfu/ml). AD 169, Towne, and Toledo, each ofwhich contain a defect in the UL131-128 locus, generated littleinfectious progeny (˜1.5×10¹ pfu/ml), consistent with their inability toefficiently express their IE 1 genes in the epithelial cells. FIX andTR, which contain wild-type UL 131-128 loci, generated intermediateyields (7×10³ and 3×10³ pfu/ml, respectively).

Example 2 Human Cytomegalovirus Virion Protein Complex is Required forEpithelial and Endothelial Cell Tropism

This example sets forth evidence demonstrating that pUL128 and pUL130form a complex with gH/gL that is incorporated into virions. The complexis required to infect endothelial and epithelial cells but notfibroblasts.

Materials and Methods

Cell Culture.

Human MRC-5 embryonic lung fibroblasts (American Type CultureCollection) were cultured in DMEM supplemented with 10% FBS, and ARPE-19retinal pigmented epithelial cells (ATCC) were cultured in DMEM/Ham'sF-12 medium (1:1) supplemented with 10% FBS. Each cell types were usedat passage 24-30.

Human umbilical vein endothelial cells (HUVEC) were obtained bycollagenase digestion of umbilical veins grown on gelatin-coated platesin RPMI 1640 supplemented with 10% FBS, endothelial cell growthsupplements (50 μg/ml, Biomedical Technologies, Stroughton, Mass.) andheparin (75 μg/ml), and used at passage 3-6. During virus adsorption,and after viral infection, HUVECs were maintained without endothelialgrowth supplements or heparin. Human foreskin fibroblasts were grown inDMEM supplemented with 10% newborn calf serum, and used at passage10-15.

Virus Preparation.

The AD169 strain of human CMV contains a frame-shift mutation in theLTL131 gene. BADwt was produced from a bacterial artificial chromosome(BAC) clone of AD 169. BADrUL131 is a derivative of BADwt in which theUL131 mutation has been repaired such that the ORF is identical to thatin the TR human CMV clinical isolate. BADdIUL131-128 is a derivative ofBADwt that was constructed by replacing the UL131-UL128 locus (basepairs 174865-176806) with a marker cassette containing the kanamycinresistance gene and LacZ gene using linear recombination. The BAC-clonedderivative of the VR1814 clinical isolate of human CMV is termed FIX,and virus reconstituted from that clone was termed BFXwt. Virus wasprepared by electroporation of BAC DNAs into fibroblasts, and the firstpassage of the virus was used in this study. Virions were partiallypurified by centrifugation through a sorbitol cushion for use as virusstocks.

Antibodies.

Anti-gB 7-17, anti-gM IMP91-3/1, anti-gH 14-4b, and AP86 monoclonalantibodies were obtained as gifts from W. Britt (University of Alabama,Birmingham). Rabbit polyclonal anti-gO antibody was obtained as a giftfrom T. Compton (University of Wisconsin, Madison). Murine monoclonalantibodies specific for pUL130 (3E3 and 3C5) and pUL128 (4B10), as wellas rabbit anti-pUL128 polyclonal antibody (R551 A), were generated byusing GST fusion proteins as immunogens.

Protein Analysis.

For pulse-chase analysis, MRC-5 cells were held for 1 h in mediumlacking methionine and cysteine beginning at 72 h post-infection at amultiplicity of three plaque-forming units per cell. 200 μCi/ml (1 Ci=37GBq) of ³⁵S Express Protein Labeling Mix (PerkinElmer) was added for 1h, the radioactivity was removed, and cells were then maintained for 20or 120 min in medium containing excess unlabeled methionine and cysteineand 10% FBS. Cells were harvested and lysed in RIPA buffer (50 mM Tris,pH 7.4/150 mM NaCl 1 mM EDTA/1% Nonidet P-40/0.1% SDS/0.5% deoxycholate)containing protease inhibitor mixture (Roche Applied Science,Indianapolis).

Before immunoprecipitation, lysates were incubated with preimmune mouseor rabbit serum overnight at 4° C., and then precleared by using proteinA Sepharose (Amersham Pharmacia Biosciences) or protein G-agarose (RocheApplied Science) to remove proteins that interact nonspecifically withthe beads. Antibodies were added to the precleared lysates, incubatedovernight at 4° C., and then protein A Sepharose or protein G-agarosewas added for 4 h at 4° C. Immune complexes were collected bycentrifugation, washed with RIPA buffer, suspended in reducing samplebuffer (50 mM Tris, pH 6.8/10% glycerol/2% SDS/1% 2-mercaptoethanol),boiled for 5 min, and proteins were separated by electrophoresis inSDS-containing polyacrylamide gels. The gM-gN complex was assayed byelectrophoresis in urea-containing polyacrylamide gels.

For analysis of virion proteins, virions were separated fromnoninfectious particles by centrifugation through glycerol-tartrategradients. The purified virions were boiled in reducing or nonreducingsample buffer, and proteins were analyzed by Western blotting.

Neutralization Assay.

Anti-pUL130 monoclonal antibodies were purified by affinitychromatography on protein G-agarose. To purify rabbit anti-pUL128polyclonal antibodies, antiserum was first passed through GST-conjugatedSepharose to deplete anti-GST antibodies that resulted from the use of apUL128-GST fusion protein as immunogen. This step was followed byaffinity purification with protein A Sepharose. Neutralization ofBADrUL131 was quantified by plaque-reduction assay. Purified antibodieswere diluted in DMEM supplemented with 5% complement inactivated FBS,and mixed with an equal volume of virus. Samples were incubated at roomtemperature for 1 h. After incubation, 300-μl aliquots (100plaque-forming units) were used to infect cell monolayers. Followingadsorption, the inoculum was removed, and cells were overlaid withmedium containing 1% agarose. Foci of GFP-expressing cells were counted2-3 weeks later. Neutralization of FIX virus was quantified by using amicroneutralization assay.

Results

pUL128 and pUL130 are Present in a Complex with gH.

To demonstrate that proteins encoded by the UL131-128 locus function incooperation with one or more virus-coded fusogenic glycoproteins, virionglycoprotein complexes were searched for UL131-UL128-coded proteins.Fibroblasts were infected with three viruses: (1) BADwt, an isolate ofthe AD169 strain of human CMV with a nonfunctional UL131 ORF; (2)BADd1UL131-128, a derivative of BADwt that lacks the UL131-UL128 locus;and (3) BADrUL131, a derivative of BADwt with a repaired UL 131 ORF. At72 h postinfection, cells were treated for 1 h with 35 5-labeledmethionine and cysteine, the label was chased for 20 or 120 min, andthen viral glycoprotein complexes were examined by immunoprecipitation(FIG. 2).

gB was found to be synthesized and glycosylated to produce a 160-kDaprotein at 20 min postlabeling, and it was partially cleaved by 120 minto generate the mature gp55-gp 116 gB complex (FIG. 2A). The gM moleculewas synthesized as a protein with an apparent molecular weight of 38kDa, and it was modified 120 min after its synthesis to migrate as adiffuse 38- to 46-kDa band (FIG. 2B). The ˜60-kDa protein thatcoprecipitated with gM was previously identified as gN (Mach M et al.(2000) J. Virol. 74:11881-92). No differences in gB or gM-gN wereobserved among the AD 169 variants.

In contrast, gH immunoprecipitates revealed distinct complexes afterinfection with the different viruses (FIG. 2C). gL and gOco-precipitated with gH from fibroblasts infected with all threeviruses. In addition, a 16-kDa protein co-precipitated from extracts ofBADwt- and BADrUL131-infected cells, but not from cells infected withBADd1UL131-128. Also, a 33-kDa protein (20 min) and a 33-plus 35-kDadoublet (120 min) were detected in the BADrUL131 gH coprecipitate. Basedon the apparent sizes, it was surmised that the 16- and 33- to 35-kDaproteins were pUL128 and pUL130. To confirm this, sequentialimmunoprecipitation assays were performed. In the immunoprecipitationassays, the gH coprecipitating proteins from BADrUL131-infected lysateswere reprecipitated with antipUL130 or -pUL128 antibodies (FIG. 2D,center and right panels). The 33- to 35- and 16-kDa proteins werespecifically precipitated with these antibodies, confirming theiridentities as pUL130 and pUL128. Antibody to gH also coprecipitated gOfrom the BADrUL131 lysates (FIG. 2D, left panel).

pLTL128-pUL130 and gO Form Separate Complexes with gH.

To further characterize the gH interactions, coimmunoprecipitationexperiments were performed using a gO-specific antibody (FIG. 3A). Thisantibody, like the gH antibody, captures the gH-gL-gO complex, and thethree components were evident in the immunoprecipitates. Neither pUL128nor pUL130 co-precipitated at a detectable level with the gO antibody,indicating that gO and pUL128-pUL130 form separate complexes with gH.Consistent with this, the pUL128-specific antibody precipitated gH, gL,and pUL130 from BADrUL131 lysates, but gO was not detected (FIG. 3B). Inaddition, anti-pUL130 antibody precipitated gH, gL, and pUL128—fromBADrUL131-infected cell lysates, but not gO (FIG. 3C). The identities ofgH and pUL 130 in the anti-pUL128 immunoprecipitate were confirmed byWestern blotting, although gO was not detected (FIG. 3D). These dataindicate that pUL128-pUL130 and gO form separate complexes with gH.

pUL128 and pUL130 are Present in Virions.

To test the possibility that the pUL128 and pUL130 complex isincorporated into virions, purified BADwt and BADrUL131 virions wereassayed by Western blotting for gH, pUL128, and pUL130 (FIG. 4A). gH wasfound to be present in both virion preparations. BADrUL131, but notBADwt virions, were found to contain pUL130, consistent with the failureof pUL130 to interact with gH in a BADwt-infected cell lysate (FIG. 2C).Notably, pUL128 also was present only in BADrUL131 virions, even thoughit interacted with gH independently of pUL130 and pUL 131 withinextracts of BADwt-infected cells (FIG. 2C). These data indicate thatonly a complete gH-gL-pUL130-pUL128 complex is incorporated intovirions.

To ascertain that pUL128 associates with gH in virions, BADrUL131 virionproteins were immunoprecipitated with pUL128-specific antibody andanalyzed by Western blotting with anti-gH or -pUL130 antibodies (FIG.4B). Both gH and pUL130 proteins were captured with anti-pUL128antibody, confirming that the three proteins are complexed in virions asin cell extracts.

Characterization of gH Complexes.

Disulfide bonds link gH to gO and gL. Based on this observation, thepossibility that gH interacts with pUL128 and pUL130 in the same mannerwas tested. BADrUL131 virion proteins were resolved by electrophoresisin reducing or nonreducing gels, transferred to membranes, and probedwith anti-pUL130 or -pUL128 antibodies. As shown in FIG. 5A, left panel,the reducing agent did not change the mobility of pUL130, suggestingthat it is not linked to other proteins through disulfide bonds. Incontrast, in the absence of 2-mercaptoethanol treatment, the anti-pUL128antibody recognized a 135-kDa protein, and treatment with the reducingagent released monomeric pUL128 (FIG. 5A, right panel). The complexpresumably includes gH and gL in addition to pUL128, because both gH andgL were precipitated from extracts of infected cells withpUL128-specific antibody (FIG. 3B). Its 135-kDa size is consistent withthe interpretation that it contains one molecule each of gH (86 kDa), gL(31 kDa), and pUL1128 (16 kDa).

Because pUL128 and gO form separate disulfide-bonded complexes with gH,the gH-gL-gO and gH-gL-pUL128 complexes present in BADwt versusBADrUL131 virions were compared. Under reducing conditions, (FIG. 5B,left panel, 132-mercaptoethanol), only monomeric, 86-kDa gH wasobserved. However, in the absence of reducing agent, more slowlymigrating bands were evident (FIG. 5B, left panel, 132-mercaptoethanol),which presumably represented disulfide bonded complexes. In BADwt, majorgH-containing complexes migrated at 300 and 220 kDa. The 220-kDa moietyis a partially modified gH-gL-gO complex, and the 300-kDa moietycorresponds to the mature gH-gL-gO (gCIII) complex (Huber M T et al.(1998) J. Virol. 72:8191-7; Huber M T et al. (1999) J. Virol.73:3886-92; and, Huber M T et al. (1997) J. Virol. 71:5391-8). Anadditional gH-containing complex was present in BADrUL131, but not BADwtvirions. The complex migrated with an apparent molecular weight of 135kDa, the same mobility as the complex recognized by antibody to pUL128(FIG. 5A), suggesting that it might be a gH-gL-pUL128 complex. MonomericgH was observed in the absence of reducing agent in virions, indicatingthat a portion of it is not covalently bonded to other glycoproteins.

Additional Western blots were carried out on the same set of virionsamples. gO-specific antibodies were observed to recognize the 300- and220-kDa complexes, but not the 135-kDa complex (FIG. 5B, center panel).In contrast, the antipUL128 antibodies reacted with the 135-kDa moietybut not the larger complexes (FIG. 5B, right panel). These data indicatethat two gH complexes are in BADrUL131 virions: gH-gL-pUL128-pUL130 andgH-gL-gO. BADwt virions contain only one gH complex, gH-gL-gO.

pUL128 and pUL130 Antibodies Block Infection of Epithelial andEndothelial Cells.

BADrUL131 virions, which contain the gHgL-pUL128-pUL130 complex, canefficiently infect endothelial cells, epithelial cells, and fibroblasts(Wang D et al. (2005) J. Virol. 79:10330-8). BADwt, which lacks thecomplex, is restricted to fibroblasts. As such, neutralization assayswere carried out to determine whether this complex is required to infectepithelial or endothelial cells.

Affinity-purified antibodies were used, and no complement was added tothe assays. As shown in FIG. 6A, pUL130-specific 3E3 monoclonal antibodyinhibited BADrUL131 infection of ARPE-19 or HUVEC cells but not MRC-5cells. Fifty percent neutralization was achieved at ˜20 μg/ml antibody.The 3C5 antibody, which recognizes a different pUL130 epitope, did notblock infection. Rabbit polyclonal antibodies to pUL128 were alsoobserved to neutralize the ability of BADrUL131 to infect ARPE-19 andHUVEC cells, but not MRC-5 cells. The patterns of inhibition were thesame for endothelial and epithelial cells, suggesting that BADrUL131utilizes the same mechanism to infect the two cell types. The antibodyto pUL128 also blocked infection of ARPE-19 and HUVEC cells by the BFXwtclinical strain of human CMV and again did not notably inhibit infectionof MRC-5 cells (FIG. 6B).

Example 3 Inhibition of Human CMV Infection of Epithelial Cells UsingAnti-CD46 Antibodies

An anti-CD46 antibody was purchased from a commercial source (antibodyJ4.48, Chemicon International). BADrUL131 is a human CMV virus that hasa functional pUL128-pUL130-containing complex and can infect bothfibroblasts and epithelial cells. A set amount of BADrUL131 was mixedwith dilutions of the antibody to CD46, and the mixtures were used toinfect ARPE-19 cells or human foreskin fibroblasts (HFF). At 24 hoursafter infection, cells were fixed and assayed for expression of thevirus coded protein, IE1, by immunofluorescence. This served as ameasure of successful infection. The percent of positive cells weredetermined and used as a measure of the efficiency of infection.

As shown in FIG. 7, an approximately 77% inhibition of human CMVinfectivity of APRE-19 epithelial cells was observed at the highestconcentration of anti-CD46 antibody tested, and this amount of antibodyhad no effect on the ability of the virus to infect control HFF cells.This indicates that the virus utilizes CD46 to enter epithelial cellsbut not for entry into fibroblasts. The inhibition of infection of theARPE-19 cells was dose-dependent, as would be expected for an antibodythat inhibits infection by binding to and blocking a receptorinteraction.

Example 4 Analysis of a Mixture of Human CMV-Specific Antibodies Usedfor Treatment of Human CMV Disease

Information set forth in this example further supports the utility ofhuman CMV UL128 and UL130 proteins (pUL128 and pUL130), and the complexwith which they are associated in virus particles, for development ofhuman CMV-specific vaccines and drugs, including antibody drugs, thatprevent or treat human CMV infection. The experiments employed acommercially available antibody preparation, CYTOGAM®, which is humanimmune globulin containing human CMV-specific antibodies. It is employedas an intravenous therapy for prophylaxis of CMV disease associated withallogeneic transplantation. There also is evidence in the literaturethat administration of such antibody preparations during pregnancy issafe and there is indication that it protects against congenitalinfection (Nigro, G et al., 2005, N. Engl. J. Med. 353:1350-62), whereapproximately 1/2000 newborns suffer moderate-to-severe consequences ofCMV infection.

The rationale for the experiment was as follows: if virion glycoproteincomplexes containing pUL128 and/or pUL130 are important for infection ofcells that are involved in human CMV pathogenesis and spread, then ahuman immune globulin preparation used to treat human CMV disease, i.e.,CYTOGAM®, should contain antibodies to one of more components of thepUL128/pUL130 complex(es), and these antibodies should neutralize humanCMV infection of cultured epithelial cells.

Initially, a western blot assay was performed to test for the presenceof antibodies specific for pUL128, pUL130 and pUL131 in CYTOGAM® (FIG.8). Partially purified glutathione-S-transferase (GST) fusion proteinswere used for the analysis (FIG. 8, top panel). Whereas antibodies inCYTOGAM® did not recognize the GST fusion partner at the level ofsensitivity employed in the experiment, the antibodies very stronglyrecognized the pUL130-containing protein and reacted to a lesser extentwith pUL128 and pUL131 fusion proteins in the western blot assay (FIG.8, bottom panel). This result indicates that CYTOGAM® containsantibodies that recognize the pUL128-pUL130-pUL131 component of thegH-gL-pUL128-pUL130-pUL131 complex in the format of a western blotassay. Further, the assay revealed that CYTOGAM® contains much morereactivity to pUL130 than to pUL128 or pUL131.

Next, gB-specific and a mixture of pUL128-pUL130-pUL131-specificantibodies were isolated from CYTOGAM® (FIG. 9). Equal amounts of IgGfrom the two preparations were then assayed for their ability toneutralize human CMV infection of epithelial cells (FIG. 10). Resultsindicated that the CYTOGAM® antibodies specific for gB and forpUL128-pUL130-pUL131 are both able to neutralize human CMV infectivity.It also suggests that at the same concentration, the partially purifiedpUL128/pUL130/pUL131 antibody has more neutralizing activity than thepartially purified gB antibody from CYTOGAM®.

To summarize, CYTOGAM® is known to contain a mixture of humanCMV-specific antibodies used for treatment of human CMV disease. We havedemonstrated that CYTOGAM® contains antibodies against thepUL128-pUL130-pUL131 complex, and these antibodies are able toneutralize the infectivity of human CMV. Indeed, it appears thatpUL128-pUL130-pUL131 antibody was more potent in the neutralizing assaythan gB antibody from CYTOGAM®. These results have implications foranti-human CMV therapy. For instance, an antibody or mixture ofantibodies that bind to pUL128-pUL130-pUl131 should be useful fortreatment of human CMV disease. Further, pUL130 appears to be aprincipal target and likely the major target within thepUL128-pUL130-pUL131 complex that is recognized by CYTOGAM®.Accordingly, an antibody or antibodies to pUL130 alone or in combinationwith antibodies to other glycoproteins may be able to substitute forCYTOGAM®. The observations with CYTOGAM® further substantiate theutility of the pUL128-pUL130-pUL131 complex and its individualcomponents as antigens for immunization. Since CYTOGAM® contains immuneglobulin from pooled human donors and since it contains predominantreactivity to the pUL130 subunit of the pUL128-pUL130-pUL131 complex,pUL130 alone or in combination with other glycoproteins should serve asan effective anti-human CMV subunit vaccine.

The present invention is not limited to the embodiments described andexemplified above, but is capable of variation and modification withinthe scope of the appended claims.

1. An immunogenic composition comprising a pharmaceutically acceptablecarrier and a complex of cytomegalovirus (CMV) proteins or fragmentsthereof, wherein the CMV proteins include each of pUL128, pUL130,pUL131, gH, gL, and gB.
 2. (canceled)
 3. The composition of claim 1,wherein multiple fragments of one or more of pUL128, pUL130, pUL131, gH,gL and gB are linked into one polypeptide chain.
 4. The composition ofclaim 3, wherein the complex is produced by expression of a CMV genomeencoding an attenuated CMV, wherein the attenuation does not affectformation of the complex.
 5. A subunit vaccine comprising apharmaceutically acceptable carrier and cytomegalovirus (CMV) proteinsor fragments thereof, wherein the CMV proteins include each of pUL128,pUL130, pUL131, gH, gL, and gB, wherein the vaccine induces an immuneresponse against CMV in a recipient.
 6. The subunit vaccine of claim 5,wherein at least one of the CMV proteins or fragments thereof is coupledto a carrier protein.
 7. The subunit vaccine of claim 5, wherein atleast one of the CMV proteins, or fragments thereof is expressed on thesurface of an attenuated CMV virus particle.
 8. The subunit vaccine ofclaim 7, wherein the at least one protein or fragment thereof is fusedto one or more other proteins or fragments thereof present on thesurface of the CMV virus particle.
 9. A nucleic acid vaccine comprisinga pharmaceutically acceptable carrier and a vector comprising at leastone nucleic acid molecule encoding cytomegalovirus (CMV) proteins orfragments thereof, wherein the CMV proteins include each of pUL128,pUL130, pUL131, gH, gL, and gB wherein the at least one nucleic acidmolecule is expressed in a vaccine recipient, and wherein the expressionproduct induces an immune response against CMV in the recipient.
 10. Thenucleic acid vaccine of claim 9, comprising a non-CMV vector. 11.-32.(canceled)
 33. A method of immunizing a patient against CMV infection byadministering to the patient the immunogenic composition of claim 1under conditions permitting the patient to develop an immune response tothe CMV proteins in the composition.
 34. (canceled)
 35. The nucleic acidvaccine of claim 9, wherein the at least one nucleic acid molecule is aDNA molecule.
 36. The nucleic acid vaccine of claim 9, wherein the atleast one nucleic acid molecule is an RNA molecule.